Methods for producing and using in vivo pseudotyped retroviruses using envelope glycoproteins from lymphocytic choriomeningitis virus (LCMV)

ABSTRACT

The present invention provides novel pseudotyped retroviral vectors that can transduce human and other cells. Vectors are provided that are packaged efficiently in packaging cells and cell lines to generate high titer recombinant virus stocks expressing novel envelope glycoproteins. The present invention further relates to compositions for gene therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application of U.S. application Ser.No. 11/593,963 filed on Nov. 7, 2006, now U.S. Pat. No. 7,439,066 whichis a Continuation application of U.S. application Ser. No. 10/993,319filed on Nov. 19, 2004 now U.S. Pat. No. 7,160,727 and issued as U.S.Pat. No. 7,160,727, which is a continuation-in-part of U.S. applicationSer. No. 10/718,262, filed Nov. 20, 2003 and issued as U.S. Pat. No.7,135,339.

U.S. GOVERNMENT RIGHTS

Portions of the present invention were made with support of the UnitedStates Government via a grant from the National Institutes of Healthunder grant numbers PPG HL-51670, DK54759, and NS34568. The U.S.Government therefore has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to improved pseudotyped retrovirus-derivedvectors useful for the expression of genes in eukaryotic cells.

BACKGROUND OF THE INVENTION

Viral vectors transduce genes into target cells with high efficienciesowing to specific virus envelope-host cell receptor interaction andviral mechanisms for gene expression. Consequently, viral vectors havebeen used as vehicles for the transfer of genes into many different celltypes. The ability to introduce and express a foreign gene in a cell isuseful for the study of gene expression and the elucidation of celllineages. Retroviral vectors, capable of integration into the cellularchromosome, have also been used for the identification ofdevelopmentally important genes via insertional mutagenesis. Viralvectors, and retroviral vectors in particular, are also used intherapeutic applications (e.g., gene therapy), in which a gene (orgenes) is added to a cell to replace a missing or defective gene due toan inherited or acquired condition or to inactivate a pathogen such as avirus.

In view of the wide variety of potential genes available for therapy, itis clear that an efficient means of delivering these genes is needed inorder treat infectious, as well as non-infectious diseases. Factorsaffecting viral vector usage include tissue tropism, stability of viruspreparations, genome packaging capacity, and construct-dependent vectorstability. In addition, in vivo application of viral vectors is oftenlimited by host immune responses against viral structural proteinsand/or transduced gene products.

Lymphocytic choriomeningitis virus was the first member of thearenavirus family identified, originally isolated in a sample from anoutbreak of St. Louis encephalitis in 1933 (Buchmeier et al.,“Arenaviridae: The Viruses and Their Replication.” In: Knipe D M, HowleyP M, eds. Fields Virology. 4^(th) ed. Philadelphia: Lippincott Williams& Wilkins, 2001:1635-1668). The virus is endemic in rodents, which serveas a reservoir. LCMV is generally noncytopathic and the most commonhuman disease associated with LCMV is aseptic meningitis. Acharacteristic feature of infection with wild type LCMV is widespreadinfection of epithelial tissues (Buchmeier et al., supra). Of interest,there is evidence to suggest that LCMV may be spread by inhalation ofinfected material [Kenyon et al., Intervirology, 33:23-31 (1992)].

SUMMARY OF THE INVENTION

The present invention provides a pseudotyped retrovirus virioncontaining an envelope glycoprotein from Lymphocytic ChoriomeningitisVirus (LCMV) strain WE54 (LCMV-WE54). LCMV-WE54 is also called LCMV-HPIin the literature. The pseudotyped retrovirus virion may contain anLCMV-WE54 envelope glycoprotein that is the same as wild type LCMV-WE54,or it may contain a variant LCMV envelope glycoprotein. A variant LCMVenvelope glycoprotein that is the same as wild type LCMV-WE54 envelopeglycoprotein except for containing slight variations in the amino acidsequence of the protein. For example, one variant LCMV-WE54 variant hasan amino acid sequence identical to the wild type protein sequenceexcept that it contains a phenylalanine (F) at position/residue 260instead of a leucine (L). In another embodiment, the LCMV-WE54 proteincontains a phenylalanine (F) at position/residue rather than a serine(S). Further, the LCMV envelope glycoprotein may have both aphenylalanine at position 260 and at position 153, i.e.,LCMV-L260F/S153F. The invention also provides further providespolynucleotide vectors that contain polynucleotides encoding anLCMV-WE54 envelope glycoprotein. The virions and vectors provided hereincan be delivered to target cells such as nervous system cells, forexample.

In one aspect, the invention thus features a method for transducing anervous system cell with a transgene. The method can include contactingthe cell with a pseudotyped retrovirus virion containing an LCMV strainWE-54 envelope glycoprotein and the transgene. The envelope glycoproteincan have a phenylalanine at residue 260, a phenylalanine at residue 153,or a phenylalanine at residue 260 and a phenylalanine at residue 153.The retrovirus virion can be a lentivirus virion (e.g., a felineimmunodeficiency virus virion).

In another aspect, the invention features a method for transducing anervous system cell with a transgene, wherein the method includescontacting the neural cell with a vector containing (a) a nucleic acidencoding an envelope glycoprotein from LCMV strain WE-54, and (b) thetransgene. The envelope glycoprotein can have a phenylalanine at residue260, a phenylalanine at residue 153, or a phenylalanine at residue 260and a phenylalanine at residue 153.

In another aspect, the invention features a method for treating a mammaldiagnosed with a neurogenetic disorder. The method can includeadministering to the mammal a pseudotyped retrovirus virion containing(a) an LCMV strain WE-54 envelope glycoprotein, and (b) a transgene. Theneurogenetic disorder can be a lysosomal storage disease, Huntington'sdisease, Parkinson's disease, amyotrophic lateral sclerosis, an ataxia,dentatorubral-pallidoluysian atrophy, prion disease, or Alzheimer'sdisease. The envelope glycoprotein can have a phenylalanine at residue260, a phenylalanine at residue 153, or a phenylalanine at residue 260and a phenylalanine at residue 153.

The invention also provides a pseudotyped feline immunodeficiency virus(FIV) virion comprising a envelope glycoprotein from LCMV envelopeglycoprotein, such as LCMV-WE54. The envelope glycoprotein may be avariant of the wild type LCMV.

The present invention further provides an isolated polynucleotide vectorcontaining a polynucleotide encoding an LCMV-WE54 envelope glycoprotein.The polynucleotide may encode the wild type LCMV-WE54 envelopeglycoprotein, or it may encode a variant LCMV envelope glycoprotein asdescribed above.

The present invention also provides a method of producing in the form ofinfectious particles a transgene vector containing a remedial gene, bytransfecting a cell with (a) a packaging vector; (b) a vector containinga nucleic acid encoding a wild type or variant LCMV envelopeglycoprotein, and (c) a transgene vector containing the remedial geneand a functional packaging signal, which by itself is incapable ofcausing a cell to produce transducing vector particles, wherein the cellproduces infectious transducing vector particles containing thetransducing transgene vector in RNA form, a Gag protein, a Pol protein,and a pseudotyped envelope glycoprotein. The packaging may be inducible.

The present invention also provides a method of delivering a remedialgene to a target cell in vivo, comprising producing viral particles bythe method described above, and then infecting the target cell with aneffective amount of the infectious transgene vector particles. Thetarget cell may be an airway epithelial cell, a central nervous systemcell, or a hepatocyte cell.

The present invention also provides a packaging cell containing anucleic acid encoding a pseudotyping LCMV envelope glycoprotein. In oneembodiment the packaging cell stably expresses a wild type LCMV-WE54. Inanother embodiment, the packaging cell stably expresses a variantenvelope glycoprotein from LCMV envelope glycoprotein, containing aphenylalanine at residue 260 and/or a phenylalanine at residue 153. Suchpackaging cells of the present invention may further contain a transgenevector, and the transgene vector may contain a remedial gene.

The present invention provides a method involving inserting a wild typeor variant LCMV envelope glycoprotein into a lipid vesicle, andelectroporating plasmid DNA into the lipid vesicle. See, for example,Yamada et al., Nature Biotech. 21:885-890 (2003).

The present invention also provides a packaging cell containing anucleic acid encoding a pseudotyping LCMV envelope glycoprotein. In oneembodiment the packaging cell stably expresses a wild type LCMV-WE54. Inanother embodiment, the packaging cell stably expresses a variantenvelope glycoprotein from LCMV envelope glycoprotein, containing aphenylalanine at residue 260 and/or a phenylalanine at residue 153. Suchpackaging cells of the present invention may further contain a transgenevector, and the transgene vector may contain a remedial gene. Thepresent invention provides a packaging cell line containing an inducibleexpression sequence that encodes a wild type or variant LCMV envelopeglycoprotein.

The present invention also provides a method of producing in the form ofinfectious particles a transducing gene transfer vector containing aremedial gene, by transfecting a packaging cell as described above witha packaging vector, and a transgene vector containing the remedial geneand a functional packaging signal, which by itself is incapable ofcausing a cell to produce transducing transgene vector particles,wherein the cell produces infectious transducing vector particlescontaining the transducing transgene vector in RNA form, a Gag protein,a Pol protein, pseudotyped with an envelope glycoprotein.

The present invention further provides a kit containing a vectorcontaining a nucleic acid encoding a wild type or variant LCMV envelopeglycoprotein; and a transgene vector containing a functional andcompatible packaging signal, the transgene vector being incapable byitself of causing a cell transfected by the transgene vector toencapsulate the RNA form of the transgene vector into a retroviralparticle containing a baculovirus envelope protein.

In one embodiment, the present invention provides a method of treatingan airway epithelial cell, wherein the airway epithelial cell has anapical surface and a basolateral surface, by administering to the apicalsurface of the airway epithelial cell a Lymphocytic ChoriomeningitisVirus (LCMV) strain WE-54 pseudotyped vector. In one embodiment, theairway epithelial cell is a human airway epithelial cell.

“Polypeptides” and “protein” are used interchangeably to refer topolymers of amino acids and do not refer to any specific lengths. Theseterms also include post-translationally modified proteins, for exampleglycosylated, acetylated, phosphorylated proteins and the like. Alsoincluded within the definition are, for example, proteins containing oneor more analogs of an amino acid (including, for example, unnaturalamino acids), proteins with substituted linkages, as well as othermodifications known in the art, both naturally occurring andnon-naturally occurring. Envelope peptides or polypeptides comprise atleast about 2, 3, 5, 10, 15, 20, 25, 30, or 50 or more consecutive aminoacid residues.

“Isolated” DNA, RNA, peptides, polypeptides, or proteins are DNA, RNA,peptides polypeptides or proteins that are isolated or purified relativeto other DNA, RNA, peptides, polypeptides, or proteins in the sourcematerial. For example, “isolated DNA” encoding the envelope protein(which would include cDNA) refers to DNA purified relative to DNA thatencodes polypeptides other than the envelope protein.

The numbering of amino acid positions within the LCMV envelopeglycoprotein of the WE54 strain is according to the numbering of GenBankAccession No. AJ318512 (designated WE-HPI). Thus, position 260 isrelative to the methionine at the first position of the sequence setforth in GenBank Accession No. AJ318512.

“Pharmaceutically acceptable” refers to molecular entities andcompositions that are physiologically tolerated and do not typicallyproduce an allergic or toxic reaction, such as gastric upset, dizzinessand the like when administered to a subject or a patient. Exemplarysubjects of the invention are vertebrates, mammals, and humans.

“Agent” herein refers to any chemical substance that causes a change.For example, agents include, but are not limited to, therapeutic genes,proteins, drugs, dyes, toxins, pharmaceutical compositions, labels,radioactive compounds, probes etc.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. LCMV WE54-FIV (n=13) transduces polarized airway epitheliapredominantly from the apical surface while VSVG (n=5) enters betterfrom the basolateral surface. Vectors were applied to the apical orbasolateral surfaces and gene transfer measured after 4 days. Thiscontrasts with the findings from the VSVG envelope (Wang et al., J.Clin. Invest. 104(11):R55-62 (1999)).

FIG. 2. Pseudotyping FIV with the LCMV-WE54 L260F mutant confersenhanced tropism human airway epithelial gene transfer. FIV expressingbeta-gal transgene was used. Enzyme activity measured by galactolightassay 4 days post-transduction.

FIG. 3. Effects of blocking agents on LCMV-FIV pseudotyped with WE54 orL260F envs. Blue bars represent control levels of beta gal in absence ofinhibitors. White bars and grey bars are laminin at 100 or 50 μg/ml,respectively. Hatched bars are IIH6 antibody. Black bar is mediacontaining alpha DG. n=2-3.

DETAILED DESCRIPTION OF THE INVENTION

Retroviruses; Retroviral Vectors

The term “retrovirus” is used in reference to RNA viruses that utilizereverse transcriptase during their replication cycle. The retroviralgenomic RNA is converted into double-stranded DNA by reversetranscriptase. This double-stranded DNA form of the virus is capable ofbeing integrated into the chromosome of the infected cell; onceintegrated, it is referred to as a “provirus.” The provirus serves as atemplate for RNA polymerase II and directs the expression of RNAmolecules that encode the structural proteins and enzymes needed toproduce new viral particles. At each end of the provirus are structurescalled “long terminal repeats” or “LTRs.” The LTR contains numerousregulatory signals including transcriptional control elements,polyadenylation signals and sequences needed for replication andintegration of the viral genome. There are several genera includedwithin the family Retroviridae, including Cisternavirus A, Oncovirus A,Oncovirus B, Oncovirus C, Oncovirus D, Lentivirus, and Spumavirus. Someof the retroviruses are oncogenic (i.e., tumorigenic), while others arenot. The oncoviruses induce sarcomas, leukemias, lymphomas, and mammarycarcinomas in susceptible species. Retroviruses infect a wide variety ofspecies, and may be transmitted both horizontally and vertically. Theyare integrated into the host DNA, and are capable of transmittingsequences of host DNA from cell to cell. This has led to the developmentof retroviruses as vectors for various purposes including gene therapy.

Retroviruses, including human foamy virus (HFV) and humanimmunodeficiency virus (HIV) have gained much recent attention, as theirtarget cells are not limited to dividing cells and their restricted hostcell tropism can be readily expanded via pseudotyping with vesicularstomatitis virus G (VSV-G) envelope glycoproteins [see, e.g., Burns etal., Proc. Natl. Acad. Sci. USA 90:8033-8037 (1993); Lever, Gene Therapy3:470-471 (1996); and Russell and Miller, J. Virol., 70:217-222 (1996)].

Vector systems generally have a DNA vector containing a small portion ofthe retroviral sequence (the viral long terminal repeat or “LTR” and thepackaging or “psi” signal) and a packaging cell line. The gene to betransferred is inserted into the DNA vector. The viral sequences presenton the DNA vector provide the signals necessary for the insertion orpackaging of the vector RNA into the viral particle and for theexpression of the inserted gene. The packaging cell line provides theviral proteins required for particle assembly [Markowitz et al., J.Virol., 62:1120 (1988)]. In one embodiment of the present invention, anFIV system employing a 3-plasmid transfection production method in 293Tcells was used [Johnston et al., J Virol. 73:4991-5000 (1999)].Replication incompetent virus was successfully produced.

The vector DNA is introduced into the packaging cell by any of a varietyof techniques (e.g., calcium phosphate coprecipitation, lipofection,electroporation). The viral proteins produced by the packaging cellmediate the insertion of the vector sequences in the form of RNA intoviral particles, which are shed into the culture supernatant.

A major limitation in the use of many commonly used retroviral vectorsin gene transfer is that many of the vectors are restricted to dividingcells. If a non-dividing cell is the target cell, then a lentivirus,which is capable of infecting non-dividing cells are provided.Alternatively, for cells that are naturally dividing or stimulated todivide by growth factors, murine leukemia virus (MLV) vectors aresuitable delivery systems.

In addition to simple retroviruses like MLV, lentiviruses can also beused as the vector. As used herein, the term “lentivirus” refers to agroup (or genus) of retroviruses that give rise to slowly developingdisease. Viruses included within this group include HIV (humanimmunodeficiency virus; including HIV type 1, and HIV type 2), theetiologic agent of the human acquired immunodeficiency syndrome (AIDS);visna-maedi, that causes encephalitis (visna) or pneumonia (maedi) insheep, the caprine arthritis-encephalitis virus, which causes immunedeficiency, arthritis, and encephalopathy in goats; equine infectiousanemia virus, which causes autoimmune hemolytic anemia, andencephalopathy in horses; feline immunodeficiency virus (FIV), whichcauses immune deficiency in cats; bovine immune deficiency virus (BIV),which causes lymphadenopathy, lymphocytosis, and possibly centralnervous system infection in cattle; and simian immunodeficiency virus(SIV), which cause immune deficiency and encephalopathy in sub-humanprimates. Diseases caused by these viruses are characterized by a longincubation period and protracted course. Usually, the viruses latentlyinfect monocytes and macrophages, from which they spread to other cells.HIV, FIV, and SIV also readily infect T lymphocytes (i.e., T-cells).

Lentiviruses including HIV, SIV, FIV and equine infectious anemia virus(EIAV) depend on several viral regulatory genes in addition to thesimple structural gag-pol-env genes for efficient intracellularreplication. Thus, lentiviruses use more complex strategies thanclassical retroviruses for gene regulation and viral replication, withthe packaging signals apparently spreading across the entire viralgenome. These additional genes display a web of regulatory functionsduring the lentiviral life cycle. For example, upon HIV-1 infection,transcription is up-regulated by the expression of Tat throughinteraction with an RNA target (TAR) in the LTR. Expression of thefull-length and spliced mRNAs is then regulated by the function of Rev,which interacts with RNA elements present in the gag region and in theenv region (RRE) [Schwartz et al. J Virol., 66:150-159 (1992)]. Nuclearexport of gag-pol and env mRNAs is dependent on the Rev function. Inaddition to these two essential regulatory genes, a list of accessorygenes, including vif, vpr, vpx, vpu, and nef, are also present in theviral genome and their effects on efficient virus production andinfectivity have been demonstrated, although they are not absolutelyrequired for virus replication [Wong-Staal and Wong-Staal, Microbiol.Rev., 55:193-205 (1991); Subbramanian and Cohen, J. Virol. 68:6831-6835(1994); and Trono, Cell 82:189-192 (1995)]. A detailed description ofthe structure of an exemplary lentivirus, HIV-1, is given in U.S. Pat.No. 6,531,123.

A “source” or “original” retrovirus is a wild-type retrovirus from whicha pseudotyped retrovirus is derived, or is used as a starting point,during construction of the packaging or transgene vector, for thepreparation of one or more of the genetic elements of the vector. Thegenetic element may be employed unchanged, or it may be mutated (but notbeyond the point where it lacks a statistically significant sequencesimilarity to the original element). A vector may have more than onesource retrovirus, and the different source retroviruses may be, e.g.,MLV, FIV, HIV-1 and HIV-2, or HIV and SIV. The term “genetic element”includes but is not limited to a gene.

A cognate retrovirus is the wild-type retrovirus with which the vectorin question has the greatest percentage sequence identity at the nucleicacid level. Normally, this will be the same as the source retrovirus.However, if a source retrovirus is extensively mutated, it isconceivable that the vector will then more closely resemble some otherretrovirus. It is not necessary that the cognate retrovirus be thephysical starting point for the construction; one may choose tosynthesize a genetic element, especially a mutant element, directly,rather than to first obtain the original element and then modify it. Theterm “cognate” may similarly be applied to a protein, gene, or geneticelement (e.g., splice donor site or packaging signal). When referring toa cognate protein, percentage sequence identities are determined at theamino acid level.

The term “cognate” retrovirus may be difficult to interpret in theextreme case, i.e., if all retroviral genetic elements have beenreplaced with surrogate non-lentiviral genetic elements. In this case,the source retrovirus strain mentioned previously is arbitrarilyconsidered to be the cognate retrovirus.

The term “replication” as used herein in reference to a virus or vector,refers not to the normal replication of proviral DNA in a chromosome asa consequence of cell reproduction, or the autonomous replication of aplasmid DNA as a result of the presence of a functional origin ofreplication. Instead “replication” refers to the completion of acomplete viral life cycle, wherein infectious viral particles containingviral RNA enter a cell, the RNA is reverse transcribed into DNA, the DNAintegrates into the host chromosome as a provirus, the infected cellproduces virion proteins and assembles them with full length viralgenomic RNA into new, equally infectious particles.

The term “replication-competent” refers to a wild-type virus or mutantvirus that is capable of replication, such that replication of the virusin an infected cell result in the production of infectious virions that,after infecting another, previously uninfected cell, causes the lattercell to likewise produce such infectious virions. The present inventioncontemplates the use of replication-defective virus.

As used herein, the term “attenuated virus” refers to any virus (e.g.,an attenuated lentivirus) that has been modified so that itspathogenicity in the intended subject is substantially reduced. Thevirus may be attenuated to the point it is nonpathogenic from a clinicalstandpoint, i.e., that subjects exposed to the virus do not exhibit astatistically significant increased level of pathology relative tocontrol subjects.

The present invention contemplates the preparation and use of a modifiedretrovirus. In some embodiments, the retrovirus is an mutant of murineleukemia virus, human immunodeficiency virus type 1, humanimmunodeficiency virus type 2, feline immunodeficiency virus, simianimmunodeficiency virus, visna-maedi, caprine arthritis-encephalitisvirus, equine infectious anemia virus, and bovine immune deficiencyvirus, or a virus comprised of portions of more than one retroviralspecies (e.g., a hybrid, comprised of portions of MLV, FIV, HIV-1 andHIV-2, or HIV-1 and/or SIV).

A reference virus is a virus whose genome is used in describing thecomponents of a mutant virus. For example, a particular genetic elementof the mutant virus may be said to differ from the cognate element ofthe reference virus by various substitutions, deletions or insertions.It is not necessary that the mutant virus actually be derived from thereference virus.

The preferred reference FIV sequence is found in Talbott et al., Proc.Natl. Acad. Sci. USA, 86:5743-7 (1989); GenBank Accession No.NC_(—)001482. In certain embodiments, a three-plasmid transienttransfection method can be used to produce replication incompetentpseudotyped retroviruses (e.g., FIV). General methods are described inWang et al., J. Clin. Invest. 104:R55-62 (1999); and Johnston et al., J.Virol. 73:4991-5000 (1999).

Retroviral Vector System

The present invention contemplates a retroviral gene amplification andtransfer system comprising a transgene vector, one or more compatiblepackaging vectors, an envelope vector, and a suitable host cell. Thevectors used may be derived from a retrovirus (e.g., a lentivirus).Retrovirus vectors allow (1) transfection of the packaging vectors andenvelope vectors into the host cell to form a packaging cell line thatproduces essentially packaging-vector-RNA-free viral particles, (2)transfection of the transgene vector into the packaging cell line, (3)the packaging of the transgene vector RNA by the packaging cell lineinto infectious viral particles, and (4) the administration of theparticles to target cells so that such cells are transduced andsubsequently express a transgene.

Either the particles are administered directly to the subject, in vivo,or the subject's cells are removed, infected in vitro with theparticles, and returned to the body of the subject.

The packaging vectors and transgene vectors of the present inventionwill generate replication-incompetent viruses. The vectors chosen forincorporation into a given vector system of the present invention aresuch that it is not possible, without further mutation of the packagingvector(s) or transgene vector, for the cotransfected cells to generate areplication-competent virus by homologous recombination of the packagingvector(s) and transgene vector alone. The envelope protein used in thepresent system can be a retroviral envelope, a synthetic or chimericenvelope, or the envelope from a non-retroviral enveloped virus (e.g.,baculovirus).

Packaging Signal

As used herein, the term “packaging signal” or “packaging sequence”refers to sequences located within the retroviral genome or a vectorthat are required for, or at least facilitate, insertion of the viral orvector RNA into the viral capsid or particle. The packaging signals inan RNA identify that RNA as one that is to be packaged into a virion.The term “packaging signal” is also used for convenience to refer to avector DNA sequence that is transcribed into a functional packagingsignal. Certain packaging signals may be part of a gene, but arerecognized in the form of RNA, rather than as a peptide moiety of theencoded protein.

The key distinction between a packaging vector and a transgene vector isthat in the packaging vector, the major packaging signal is inactivated,and, in the transgene vector, the major packaging signal is functional.Ideally, in the packaging vector, all packaging signals would beinactivated, and, in the transgene vector, all packaging signals wouldbe functional, However, countervailing considerations, such asmaximizing viral titer, or inhibiting homologous recombination, may lendsuch constructs less desirable.

Packaging System; Packaging Vectors; Packaging Cell Line

A packaging system is a vector, or a plurality of vectors, whichcollectively provide in expressible form all of the genetic informationrequired to produce a virion that can encapsidate suitable RNA,transport it from the virion-producing cell, transmit it to a targetcell, and, in the target cell, cause the RNA to be reverse transcribedand integrated into the host genome in a such a manner that a transgeneincorporated into the aforementioned RNA can be expressed. However, thepackaging system must be substantially incapable of packaging itself.Rather, it packages a separate transgene vector.

In the present invention, the packaging vector will provide functionalequivalents of the gag and pol genes (a “GP” vector). The env gene(s)will be provided by the envelope vector. In theory, a three vectorsystem (“G”, “P”, and “E” vectors) is possible if one is willing toconstruct distinct gag and pol genes on separate vectors, and operablylink them to different regulatable promoters (or one to a regulatableand the other to a constitutive promoter) such that their relativelevels of expression can be adjusted appropriately.

A packaging cell line is a suitable host cell transfected by a packagingsystem that, under achievable conditions, produces viral particles. Asused herein, the term “packaging cell lines” is typically used inreference to cell lines that express viral structural proteins (e.g.,gag, pol and env), but do not contain a packaging signal. For example, acell line has been genetically engineered to carry at one chromosomalsite within its genome, a 5′-LTR-gag-pol-3′-LTR fragment that lacks afunctional psi⁺ sequence (designated as Δ-psi), and a 5′-LTR-env-3′-LTRfragment that is also Δ-psi located at another chromosomal site. Whileboth of these segments are transcribed constitutively, because the psi⁺region is missing and the viral RNA molecules produced are less thanfull-size, empty viral particles are formed.

If a host cell is transfected by the packaging vector(s) alone, itproduces substantially only viral particles without the full-lengthpackaging vector. In one example, less than 10% of the viral particlesproduced by the packaging cell contain full length packagingvector-derived RNA. However, since the packaging vector lacks afunctional primer binding site, even if these particles infect a newcell, the packaging vector RNA will not be reverse transcribed back intoDNA and therefore the new cell will not produce virion. Thus, by itself,the packaging vector is a replication-incompetent virus.

In some embodiments, the packaging cell and/or cell line contains atransgene vector. The packaging cell line will package the transgenevector into infectious particles. Such a cell line is referred to hereinas a “transgenic virion production cell line.”

It is contemplated that packaging may be inducible, as well asnon-inducible. In inducible packaging cells and packaging cell lines,retroviral particles are produced in response to at least one inducer.In non-inducible packaging cell lines and packaging cells, no inducer isrequired in order for retroviral particle production to occur.

The packaging vectors necessarily differ from wild-type,replication-competent retroviral genomes by virtue of the inactivationof at least one packaging signal of the cognate wild-type genome. Morethan one packaging signal may be inactivated. In one example, only theretroviral genes provided by the packaging vector are those encodingstructural, or essential regulatory, proteins.

Envelope Protein Vectors

The envelope proteins encoded by the packaging vector are viralproteins. The vector containing an envelope protein that is differentfrom the packaging virus genes is commonly referred to as an envelopepseudotyping vector.

Env glycoproteins: The Env glycoproteins of a retrovirus may be replacedwith Env glycoproteins of other retroviruses, of non-retroviral viruses,or with chimeras of these glycoproteins with other peptides or proteins.An example of a non-lentiviral envelope glycoprotein of interest is thelymphocytic choriomeningitis virus (LCMV) strain WE54 envelopeglycoprotein. These envelope glycoproteins increase the range of cellsthat can be transduced with retroviral derived vectors. In one exampleof the present invention, the envelope protein is a “mutant” or variantLCMV-WE54 protein, such as a LCMV envelope glycoprotein containing asingle point mutation (L260F). In another embodiment, the variant LCMVenvelope glycoprotein contains a single point mutation (S153F). Inanother embodiment, the variant LCMV envelope glycoprotein contains twopoint mutations L260F and S153. Such mutations allow further specificityas to what cell types the pseudotyped virus will infect, i.e., theaffinity of the vector is modified.

As used herein, the LCMV-WE54 envelope glycoproteins include variants orbiologically active fragments of the proteins. A “variant” of theprotein is a protein that is not completely identical to a nativeprotein. A variant protein can be obtained by altering the amino acidsequence by insertion, deletion or substitution of one or more aminoacid. The amino acid sequence of the protein is modified, for example bysubstitution, to create a polypeptide having substantially the same orimproved qualities as compared to the native polypeptide. Thesubstitution may be a conserved substitution. A “conserved substitution”is a substitution of an amino acid with another amino acid having asimilar side chain. A conserved substitution would be a substitutionwith an amino acid that makes the smallest change possible in the chargeof the amino acid or size of the side chain of the amino acid(alternatively, in the size, charge or kind of chemical group within theside chain) such that the overall peptide retains its spatialconformation but has altered biological activity. For example, commonconserved changes might be Asp to Glu, Asn or Gln; His to Lys, Arg orPhe; Asn to Gln, Asp or Glu and Ser to Cys, Thr or Gly. Alanine iscommonly used to substitute for other amino acids. The 20 essentialamino acids can be grouped as follows: alanine, valine, leucine,isoleucine, proline, phenylalanine, tryptophan and methionine havingnonpolar side chains; glycine, serine, threonine, cysteine, tyrosine,asparagine and glutamine having uncharged polar side chains; aspartateand glutamate having acidic side chains; and lysine, arginine, andhistidine having basic side chains (Stryer, Biochemistry (2^(nd) ed.),W. H. Freeman and Co., San Francisco (1981) pp. 14-15; Lehninger,Biochemistry (2^(nd) ed.), Institute of Electrical & Electronics Enginee(1975), pp. 73-75).

It is known that variant polypeptides can be obtained based onsubstituting certain amino acids for other amino acids in thepolypeptide structure in order to modify or improve biological activity.For example, through substitution of alternative amino acids, smallconformational changes may be conferred upon a polypeptide that resultin increased bioactivity. One can use the hydropathic index of aminoacids in conferring interactive biological function on a polypeptide,wherein it is found that certain amino acids may be substituted forother amino acids having similar hydropathic indices and still retain asimilar biological activity.

A variant of the invention may include amino acid residues not presentin the corresponding native protein, or may include deletions relativeto the corresponding native protein. A variant may also be a truncatedfragment as compared to the corresponding native protein, i.e., only aportion of a full-length protein. Protein variants also include peptideshaving at least one D-amino acid.

The LCMV-WE54 envelope glycoprotein of the present invention may beexpressed from isolated nucleic acid (DNA or RNA) sequences encoding theproteins. Amino acid changes from the native to the variant protein maybe achieved by changing the codons of the corresponding nucleic acidsequence. Recombinant is defined as a peptide or nucleic acid producedby the processes of genetic engineering. It should be noted that it iswell-known in the art that, due to the redundancy in the genetic code,individual nucleotides can be readily exchanged in a codon, and stillresult in an identical amino acid sequence.

The starting material (such as a gene encoding an LCMV-WE54 envelopeglycoprotein) used to make the complexes of the present invention may besubstantially identical to wild-type genes, or may be variants of thewild-type gene. Further, the polypeptide encoded by the startingmaterial may be substantially identical to that encoded by the wild-typegene, or may be a variant of the wild-type gene. The following terms areused to describe the sequence relationships between two or more nucleicacids or polynucleotides: (a) “reference sequence,” (b) “comparisonwindow,” (c) “sequence identity,” (d) “percentage of sequence identity,”and (e) “substantial identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may include additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot include additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm. Preferred,non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller, CABIOS, 4:11 (1988); the local homology algorithmof Smith et al., Adv. Appl. Math., 2:482 (1981); the homology alignmentalgorithm of Needleman and Wunsch, J. Mol. Biol., 48:443-453 (1970); thesearch-for-similarity-method of Pearson and Lipman, Proc. Natl. Acad.Sci. USA, 85:2444 (1988); the algorithm of Karlin and Altschul, Proc.Natl. Acad. Sci. USA, 87:2264 (1990), modified as in Karlin andAltschul, Proc. Natl. Acad. Sci. USA, 90:5873 (1993).

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Version 8 (availablefrom Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.,Gene, 73:237 (1988); Higgins et al., CABIOS, 5:151 (1989); Corpet etal., Nucl. Acids Res., 16:10881 (1988); Huang et al., CABIOS, 8:155(1992); and Pearson et al., Meth. Mol. Biol., 24:307 (1994). The ALIGNprogram is based on the algorithm of Myers and Miller, supra. The BLASTprograms of Altschul et al., J. Mol. Biol., 215:403 (1990); Nucl. AcidsRes., 25:3389 (1990), are based on the algorithm of Karlin and Altschulsupra.

Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (World Wide Web at “ncbi”dot “nlm” dot “nih” dot “gov”). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold. These initial neighborhood word hits act as seedsfor initiating searches to find longer HSPs containing them. The wordhits are then extended in both directions along each sequence for as faras the cumulative alignment score can be increased. Cumulative scoresare calculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always>0) and N (penalty scorefor mismatching residues; always<0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when the cumulative alignment scorefalls off by the quantity X from its maximum achieved value, thecumulative score goes to zero or below due to the accumulation of one ormore negative-scoring residue alignments, or the end of either sequenceis reached.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences. One measure of similarity provided by the BLAST algorithmis the smallest sum probability (P(N)), which provides an indication ofthe probability by which a match between two nucleotide or amino acidsequences would occur by chance. For example, a test nucleic acidsequence is considered similar to a reference sequence if the smallestsum probability in a comparison of the test nucleic acid sequence to thereference nucleic acid sequence is less than about 0.1, more preferablyless than about 0.01, and most preferably less than about 0.001.

To obtain gapped alignments for comparison purposes, Gapped BLAST (inBLAST 2.0) can be utilized as described in Altschul et al., Nucl. AcidsRes. 25:3389 (1997). Alternatively, PSI-BLAST (in BLAST 2.0) can be usedto perform an iterated search that detects distant relationships betweenmolecules. See Altschul et al., supra. When utilizing BLAST, GappedBLAST, PSI-BLAST, the default parameters of the respective programs (e.gBLASTN for nucleotide sequences, BLASTX for proteins) can be used. TheBLASTN program (for nucleotide sequences) uses as defaults a wordlength(W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix. See World Wide Web at “ncbi” dot “nlm” dot“nih” dot “gov.” Alignment may also be performed manually by inspection.

For purposes of the present invention, comparison of nucleotidesequences for determination of percent sequence identity to the promotersequences disclosed herein is made using the BlastN program (version1.4.7 or later) with its default parameters or any equivalent program.By “equivalent program” is intended any sequence comparison programthat, for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by the preferred program.

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to a specifiedpercentage of residues in the two sequences that are the same whenaligned for maximum correspondence over a specified comparison window,as measured by sequence comparison algorithms or by visual inspection.When percentage of sequence identity is used in reference to proteins itis recognized that residue positions which are not identical oftendiffer by conservative amino acid substitutions, where amino acidresidues are substituted for other amino acid residues with similarchemical properties (e.g., charge or hydrophobicity) and therefore donot change the functional properties of the molecule. When sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity.” Means for makingthis adjustment are well known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may include additions or deletions (i.e., gaps) ascompared to the reference sequence (which does not include additions ordeletions) for optimal alignment of the two sequences. The percentage iscalculated by determining the number of positions at which the identicalnucleic acid base or amino acid residue occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparison,and multiplying the result by 100 to yield the percentage of sequenceidentity.

(e)(i) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide includes a sequence that has at least 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably at least 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%,91%, 92%, 93%, or 94%, and most preferably at least 95%, 96%, 97%, 98%,or 99% sequence identity, compared to a reference sequence using one ofthe alignment programs described using standard parameters. One of skillin the art will recognize that these values can be appropriatelyadjusted to determine corresponding identity of proteins encoded by twonucleotide sequences by taking into account codon degeneracy, amino acidsimilarity, reading frame positioning, and the like. Substantialidentity of amino acid sequences for these purposes normally meanssequence identity of at least 70%, more preferably at least 80%, 90%,and most preferably at least 95%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions(see below). Generally, stringent conditions are selected to be about 5EC lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. However, stringentconditions encompass temperatures in the range of about 1 EC to about 20EC, depending upon the desired degree of stringency as otherwisequalified herein. Nucleic acids that do not hybridize to each otherunder stringent conditions are still substantially identical if thepolypeptides they encode are substantially identical. This may occur,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code. One indication that twonucleic acid sequences are substantially identical is when thepolypeptide encoded by the first nucleic acid is immunologically crossreactive with the polypeptide encoded by the second nucleic acid.

(e)(ii) The term “substantial identity” in the context of a peptideindicates that a peptide includes a sequence with at least 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%, 91%,92%, 93%, or 94%, or even more preferably, 95%, 96%, 97%, 98% or 99%,sequence identity to the reference sequence over a specified comparisonwindow. Preferably, optimal alignment is conducted using the homologyalignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48:443(1970). An indication that two peptide sequences are substantiallyidentical is that one peptide is immunologically reactive withantibodies raised against the second peptide. Thus, a peptide issubstantially identical to a second peptide, for example, where the twopeptides differ only by a conservative substitution.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

As noted above, another indication that two nucleic acid sequences aresubstantially identical is that the two molecules hybridize to eachother under stringent conditions. The phrase “hybridizing specificallyto” refers to the binding, duplexing, or hybridizing of a molecule onlyto a particular nucleotide sequence under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA. Bind(s) substantially refers to complementary hybridization betweena probe nucleic acid and a target nucleic acid and embraces minormismatches that can be accommodated by reducing the stringency of thehybridization media to achieve the desired detection of the targetnucleic acid sequence.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridizations are sequence dependent, andare different under different environmental parameters. Longer sequenceshybridize specifically at higher temperatures. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Specificity istypically the function of post-hybridization washes, the criticalfactors being the ionic strength and temperature of the final washsolution. For DNA-DNA hybrids, the T_(m) can be approximated from theequation of Meinkoth and Wahl, Anal. Biochem., 138:267 (1984); T_(m)81.5° C.+16.6 (log M)+0.41 (% GC) −0.61 (% form) −500/L; where M is themolarity of monovalent cations, % GC is the percentage of guanosine andcytosine nucleotides in the DNA, % form is the percentage of formamidein the hybridization solution, and L is the length of the hybrid in basepairs. T_(m) is reduced by about 1 EC for each 1% of mismatching; thus,T_(m), hybridization, and/or wash conditions can be adjusted tohybridize to sequences of the desired identity. For example, ifsequences with >90% identity are sought, the T_(m) can be decreased 10EC. Generally, stringent conditions are selected to be about 5 EC lowerthan the thermal melting point (T_(m)) for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3,or 4 EC lower than the thermal melting point (T_(m)); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9, or 10 EC lower than the thermal melting point (T_(m)); low stringencyconditions can utilize a hybridization and/or wash at 11, 12, 13, 14,15, or 20 EC lower than the thermal melting point (T_(m)). Using theequation, hybridization and wash compositions, and desired T, those ofordinary skill will understand that variations in the stringency ofhybridization and/or wash solutions are inherently described. If thedesired degree of mismatching results in a T of less than 45 EC (aqueoussolution) or 32 EC (formamide solution), it is preferred to increase theSSC concentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen,Laboratory Techniques in Biochemistry and Molecular BiologyHybridization with Nucleic Acid Probes, part I chapter 2 “Overview ofprinciples of hybridization and the strategy of nucleic acid probeassays” Elsevier, N.Y. (1993). Generally, highly stringent hybridizationand wash conditions are selected to be about 5° C. lower than thethermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH.

An example of highly stringent wash conditions is 0.15 M NaCl at 72° C.for about 15 minutes. An example of stringent wash conditions is a0.2×SSC wash at 65° C. for 15 minutes [see, Sambrook et al., MolecularCloning: A Laboratory Manual (3^(rd) ed.), Cold Spring Harbor LaboratoryPress, (2001) for a description of SSC buffer]. Often, a high stringencywash is preceded by a low stringency wash to remove background probesignal. An example medium stringency wash for a duplex of, e.g., morethan 100 nucleotides, is 1×SSC at 45° C. for 15 minutes. An example lowstringency wash for a duplex of, e.g., more than 100 nucleotides, is4-6×SSC at 40° C. for 15 minutes. For short probes (e.g., about 10 to 50nucleotides), stringent conditions typically involve salt concentrationsof less than about 1.5 M, more preferably about 0.01 to 1.0 M, Na ionconcentration (or other salts) at pH 7.0 to 8.3, and the temperature istypically at least about 30° C. and at least about 60° C. for longprobes (e.g., >50 nucleotides). Stringent conditions may also beachieved with the addition of destabilizing agents such as formamide. Ingeneral, a signal to noise ratio of 2× (or higher) than that observedfor an unrelated probe in the particular hybridization assay indicatesdetection of a specific hybridization. Nucleic acids that do nothybridize to each other under stringent conditions are stillsubstantially identical if the proteins that they encode aresubstantially identical. This occurs, e.g., when a copy of a nucleicacid is created using the maximum codon degeneracy permitted by thegenetic code.

Very stringent conditions are selected to be equal to the T_(m) for aparticular probe. An example of stringent conditions for hybridizationof complementary nucleic acids that have more than 100 complementaryresidues on a filter in a Southern or Northern blot is 50% formamide,e.g., hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37 EC, and awash in 0.1×SSC at 60 to 65 EC. Exemplary low stringency conditionsinclude hybridization with a buffer solution of 30 to 35% formamide, 1 MNaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C.Exemplary moderate stringency conditions include hybridization in 40 to45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSCat 55 to 60° C.

Chimeric Env Glycoproteins: A chimera may be constructed of an envglycoprotein and of a ligand that binds to a specific cell surfacereceptor in order to target the vector to cells expressing thatreceptor. Examples are chimeras including FLA16 (a 6 amino acid peptidethat binds integrin receptors), erythropoietin (which binds theerythropoietin receptor), human heregulin (which binds the EGF andrelated receptors). Alternatively, the chimera could include an antibodyvariable light or heavy domain, or both domains joined by suitablepeptide linker (a so-called single chain antibody). Such an antibodydomain could target any desired cell surface molecule, such as a tumorantigen, the human low-density lipoprotein receptor, or a determinant onhuman MHC Class I molecules.

Derivatized Env Glycoproteins: Virions may be chemically, enzymaticallyor physically modified after production in order to alter their cellspecificity. Examples of modifications include chemical or enzymaticaddition of a ligand which would be recognized by a cell surfacereceptor (e.g., addition of lactose so that the virions will transducehuman hepatoma cells which express asialoglycoprotein receptors), orincubation of the virus with a biotinylated antibody directed againstthe vector's Env protein, followed by addition of a streptavidin-linkedligand recognized by the cell-surface receptor. A heterobispecificantibody also could be used to link the virion's Env protein to such aligand.

Transgene Vectors

A transgene vector is an expression vector that bears an expressiblenonretroviral gene of interest and includes at least one functionalretroviral packaging signal, so that, after the transgene vector istransfected into a packaging cell line, the transgene vector istranscribed into RNA, and this RNA is packaged into an infectious viralparticle. These particles, in turn, infect target cells, their RNA isreverse transcribed into DNA, and the DNA is incorporated into the hostcell genome as a proviral element, thereby transmitting the gene ofinterest to the target cells.

As used herein, the term “transduction” refers to the delivery of agene(s) using a viral or retroviral vector by means of infection ratherthan by transfection. In certain embodiments, retroviral vectors aretransduced. Thus, a “transduced gene” is a gene that has been introducedinto the cell via retroviral or vector infection and provirusintegration. In certain embodiments, viral vectors (e.g., “transgenevectors”) transduce genes into “target cells” or host cells. The,present invention encompasses transgene vectors that are suitable foruse in the present invention that are linked to any gene of interest (ora “marker gene” or “reporter gene,” used to indicate infection orexpression of a gene).

As used herein, the term “long-term transduction” refers to vectors thatare capable of remaining transduced in host or target cells for timeperiods that are longer than those observed with other vectors. Forexample, the present invention provides retroviral vectors that arecapable of remaining transduced for at least 120 days, at least oneyear, or for the life of the subject or the necessary time course oftreatment. The duration of expression is a function of the choice ofpromoter and the target cell type, more so than the choice of vector.

The term “stable transduction” or “stably transduced” refers to theintroduction and integration of foreign DNA into the genome of thetransducted cell. The term “stable transductant” refers to a cell thathas stably integrated foreign DNA into the genomic DNA.

The term “transient transduction” or “transiently transduced” refers tothe introduction of foreign DNA into a cell where the foreign DNA failsto integrate into the genome of the transducted cell. The foreign DNApersists in the nucleus of the transducted cell for several days. Duringthis time the foreign DNA is subject to the regulatory controls thatgovern the expression of endogenous genes in the chromosomes. The term“transient transductant” refers to cells that have taken up foreign DNAbut have failed to integrate this DNA.

In some embodiments, the target and/or host cells of the presentinvention are “non-dividing” cells. These cells include cells such asneuronal cells that do not normally divide. However, it is not intendedthat the present invention be limited to non-dividing cells (including,but not limited to muscle cells, white blood cells, spleen cells, livercells, eye cells, epithelial cells).

In some embodiments, the vector and the vector progeny are capable oftransducing a plurality of target cells so as to achieve vector titersof at least 10⁵ cfu/ml. The multiplicity of infection (MOI) may be atleast one (i.e., one hit on average per cell), or even at least two.

Transgene

The transgene is a gene encoding a polypeptide that is foreign to theretrovirus(es) from which the vector is primarily derived, and has auseful biological activity in the organism that is ultimately infectedwith the transgene vector in its virion-packaged form.

The transgene may be identical to a wild-type gene, or it may containone or more mutations. The transgene may be derived from genomic DNA,cDNA, synthetic DNA, or a combination thereof. Intronless “minigenes,”which are normal genes from which introns have been removed, have beenespecially popular. Intron-containing genes may be employed, but theymay be inserted into the vector in the reverse orientation if removal ofthe introns is not desired. Silent mutations may be introduced tofacilitate gene manipulation, to avoid undesirable secondary structurein the mRNA, to inhibit recombination, to control splicing, etc.Non-silent mutations alter the encoded protein, and may be eithergratuitous, or aimed at beneficially altering the biological activity ofthe protein.

One example of a transgene is a remedial gene. As used herein, the term“remedial gene” refers to a gene whose expression is desired in a cellto correct an error in cellular metabolism, to inactivate a pathogen orto kill a cancerous cell. For example, the adenosine deaminase (ADA)gene is the remedial gene when carried on a retroviral vector used tocorrect ADA deficiency in a patient.

The applications of transgenes include the following:

cell marking: for some purposes, it is useful to follow cells after theyhave been introduced into a patient.

anti-pathogen or anti-parasite: anti-pathogen genes or anti-parasite canbe introduced into a host infested, or especially vulnerable toinfestation, by the pathogen or parasite in question.

genetic disease: an inherited genetic defect may be ameliorated bysupplying a functional gene.

It is not necessary that the endogenous gene be repaired by homologousrecombination. Monogenetic genetic diseases are of particular interest.Suitable approaches include providing genes encoding the enzyme ADA,especially to hematopoietic stem cells so as to provide long termtreatment of ADA deficiency; and correcting familialhypercholesterolemia with a vector encoding the low density lipoprotein(LDL) receptor.

Gene therapy has been used to successfully correct inborn errors ofmetabolism using existing vector systems. For example, the adenosinedeaminase gene has been introduced into peripheral blood lymphocytes andcord blood stem cells via retroviral vectors in order to treat patientswith severe combined immunodeficiency due to a lack of functionaladenosine deaminase [Culver et al., Human Gene Ther., 2:107 (1991)].Partial correction of familial hypercholesterolemia has been achievedusing existing retroviral vectors to transfer the receptor for lowdensity lipoproteins (LDL) into hepatocytes. However, it was estimatedthat only 5% of the liver cells exposed to the recombinant virusincorporated the LDL receptor gene with the vector utilized [Grossman etal., Nat. Genet., 6:335 (1994)].

A number of single-gene disorders have been targeted for correctionusing gene therapy. These disorders include hemophilia (lack of FactorVIII or Factor IX), cystic fibrosis (lack of cystic fibrosistransmembrane conductance regulator), emphysema (defectiveα-1-antitrypsin), thalassemia and sickle cell anemia (defectivesynthesis of β-globin), phenylketonuria (deficient phenylalaninehydroxylase) and muscular dystrophy (defective dystrophin) [for reviewsee Miller, Nature 357:455 (1992)]. Human gene transfer trials have beenapproved for a number of these diseases.

The molecular genetics of cystic fibrosis (CF) has been studied andgradually understood in recent years. Many CF patients carry a singleamino acid deletion ((F508) mutation in one of the twonucleotide-binding domains in the CF transmembrane regulator (CFTR)protein. Other forms of genetic mutations in the CFTR genes have alsobeen identified. This rich genetic information makes CF an ideal genetherapy candidate.

The target cells for CF patients are undifferentiated, proliferating anddifferentiated, non-proliferating lung epithelial cells. For example,both the dividing and non-dividing lung epithelial cell types can betargeted by pseudotyped retroviral vectors carrying a wild type CFTRcDNA.

CF patients have CFTR mutations that leads to basic chloride flux defectin the respiratory ciliated epithelial cells. This CFTR dysfunctioncauses chronic infection and inflammation of the respiratory tract andleads to high morbidity and mortality in CF patients. The CFTR cDNA genetransfer by adenoviral vectors or liposomes has demonstrated partialcorrection of the defective CFTR channel activity in the nasalepithelium of CF patients. Recent studies suggest that gene therapy mayoffer great benefits to CF patients even if only partial correction ofCFTR gene function is achieved.

In some embodiments, a neurogenetic or neurodegenerative (i.e., adegenerative or inherited disorder of the nervous system) disorder canbe treated by contacting a cell or administering to a subject (e.g., amammal) a transgene. The transgene can be contained within a virion(e.g., a pseudotyped retrovirus virion containing a LCMV strain WE-54envelope glycoprotein and the transgene), or a vector (e.g., a vectorcontaining (a) a nucleic acid that encodes an envelope glycoprotein fromLymphocytic Choriomeningitis Virus strain WE-54, and (b) the transgene).Any suitable method can be used to contact a cell (e.g., a neural cellfrom an area of the central nervous system such as the ependyma,choroids plexus, subventricular zone (SVZ), rostral migratory stream(RMS), or olfactory bulb (OB)). The cell can be contacted in vitro, exvivo, or in vivo, such as when a subject is directed treated with avirion or a vector as described herein. Further, any suitable method canbe used to administer a virion or a vector to a subject diagnosed with aneurogenetic disorder. Neurogenetic disorders that can be treated usingsuch methods include, without limitation, lysosomal storage diseases(e.g., leukodystrophies, mucopolysaccharidoses, and ceroidlipofuscinoses), Huntington's disease, Parkinson's disease, amyotrophiclateral sclerosis, ataxias, dentatorubral-pallidoluysian atrophy, priondisease, and Alzheimer's disease.

cancer: cancers may be treated with vectors carrying genes that expresscancer antigens, or immunomodulatory proteins, and thereby stimulate animmune response against the cancer cells, or express a normal tumorsuppressor gene to replace the function of a mutated, tumor-prone gene,such as a p53 mutant.

In addition to replacement of defective genes, it has been proposed thatviral vectors could be used to deliver genes designed to stimulateimmunity against or to otherwise destroy tumor cells. Although theintegration of therapeutic genes into tumor cells is not required forcancer gene therapy application in most cases, sustained expression ofthe therapeutic genes in tumor cells may be required, for example, toelicit a long lasting in vivo anti-tumor immunity.

Gene therapy, originally developed for treating inherited and acquireddiseases by introducing therapeutic genes to somatic cells, has greatpotential for cancer treatment. There are three major components to beconsidered in the design and development of a gene therapy regimen: thetherapeutic genes, the mode of gene delivery (ex vivo or in vivo), andan appropriate preclinical study model for the assessment of thetherapeutic efficacy. Various therapeutic genes have been utilized incancer treatments. The common examples include: (1) genes that arecapable of changing the cellular sensitivity to chemo-or radiationtherapy in cancer patients either to sensitize tumor cells, or tominimize the damage of chemotherapy to normal cells such as thehematopoietic stem cells, (2) genes that interfere with proliferatingtumor cell cycle by either replacing the mutated genes (i.e., tumorsuppresser genes and apoptotic genes), or inactivating the oncogenes toprevent further tumor development, and (3) genes that can augment asystemic anti-tumor immunity in cancer patients; this can beaccomplished by the injection of modified tumor infiltrating lymphocytes(TIL) or immunomodulatory gene-modified tumor cells, or by themodification of antigen presenting cells (APC). Retroviral vectorscontaining genes encoding tumor necrosis factor (TNF) or interleukin-2(IL-2) have been transferred into tumor-infiltrating lymphocytes inpatients [Kasid et al., Proc. Natl. Acad. Sci. USA. 87:473-477 (1990);and Rosenberg, Human Gene Therapy 5:140 (1994)]. It is postulated thatthe secretion of TNF or IL-2 stimulates a tumor-specific immune responseresulting in the destruction of the tumor or the recruitment ofeffective tumor infiltrating lymphocytes from nearby lymph nodes. Otherproposed anti-tumor gene therapy strategies include the delivery oftoxin genes to the tumor cell.

Applications of antisense genes or oligonucleotides in inhibition ofoncogenes and modulation of growth factors have the potential to reducethe mortality of cancer, in particular, human leukemia [For review see,Gewirtz, Stem Cells 3:96 (1993); and Neckers and Whitesell, Amer. J.Physiol., 265:L1 (1993)].

HIV: vectors may be used to deliver transgenes that protect susceptiblecells against HIV by synthesizing proteins, antisense RNAs, or ribozymesthat block HIV binding and entry, reverse transcription, integration, orreplication. Of course, the transgenes must be regulated so they do notinterfere with the packaging of the transgene vector.

Selectable and Screenable Markers

A vector may contain one or more selectable or screenable markers. Suchmarkers are typically used to determine whether the vector has beensuccessfully introduced into a host or target cell. A selectable markeris a gene whose expression substantially affects whether a cell willsurvive under particular controllable conditions. A selectable markermay provide for positive selection (cells with the marker are morelikely to survive), negative selection (cells with the marker are lesslikely to survive), or both (the choice of environmental conditiondictating whether positive or negative selection occurs).

Selectable markers include those that confer antibiotic resistance (orsensitivity), the ability to utilize a particular nutrient, andresistance (or sensitivity) to high (or low) temperature. Suitableselectable markers include the bacterial neomycin and hygromycinphosphotransferase resistance genes, which confers resistance to G418and hygromycin, respectively, the bacterial gpt gene, which allows cellsto grow in a medium containing mycophenolic acid, xanthine andaminopterin; the bacterial hisD gene that allows cells to grow in amedium lacking histidine but containing histidinol; the multidrugresistance gene mdr; the hprt and HSV thymidine kinase genes, whichallow otherwise hprt-or tk-cells to grow in a medium containinghypoxanthine, amethopterin and thymidine, and the bacterial genesconferring resistance to puromycin or phleomycin. Positive or negativeselection may require the use of a particular strain of host cell forthe selection to be effective.

Screenable markers are genes that encode a product whose presence isreadily detectable, directly or indirectly, but do not necessarilyaffect cell survival. The green fluorescent protein (GFP) is an example.Any cell surface protein not native to the host cell can be used as animmunoscreenable marker. Transformed cells may be segregated out byusing a fluorescent antibody to the protein and a cell sorter. Manyenzyme-encoding genes are useful as screenable markers, especially thoseencoding enzymes that can act upon a substrate to provide a colored orluminescent product. The luciferase and beta-galactosidase genes havebeen especially popular.

A dominant marker encodes an activity that can be detected in anyeukaryotic cell line. Examples of dominant selectable markers includethe bacterial aminoglycoside 3′ phosphotransferase gene (also referredto as the neo gene) that confers resistance to the drug G418 inmammalian cells, the bacterial hygromycin G phosphotransferase (hyg)gene that confers resistance to the antibiotic hygromycin and thebacterial xanthine-guanine phosphoribosyl transferase gene (alsoreferred to as the gpt gene) that confers the ability to grow in thepresence of mycophenolic acid. Other selectable markers are not dominantin that their use must be in conjunction with a cell line that lacks therelevant activity. Examples of non-dominant selectable markers includethe thymidine kinase (tk) gene that is used in conjunction with tk celllines, the CAD gene that is used in conjunction with CAD-deficient cellsand the mammalian hypoxanthine-guanine phosphoribosyl transferase (hprt)gene that is used in conjunction with hprt-cell lines.

A review of the use of markers in mammalian cell lines is provided inSambrook, et al., Molecular Cloning: A Laboratory Manual (2^(nd) ed.),Cold Spring Harbor Laboratory Press, New York (1989) pp. 16.9-16.15.

Regulation of Gene Expression

The transgene(s) of the transgene vector, and the marker(s) and viralgenes (or replacements) of the packaging and transgene vectors, and theglycoprotein genes of the envelope vector are expressed under thecontrol of regulatory elements.

As used herein, the term “regulatory element” refers to a geneticelement that controls some aspect of the expression of nucleic acidsequences. For example, a promoter is a regulatory element thatfacilitates the initiation of transcription of an operably linked codingregion. Other regulatory elements are splicing signals, polyadenylationsignals, termination signals, etc. A constitutive promoter is one thatis always active at essentially a constant level.

Transcriptional control signals in eukaryotes comprise “promoter” and“enhancer” elements. Promoters and enhancers consist of short arrays ofDNA sequences that interact specifically with cellular proteins involvedin transcription [Maniatis et al., Science 236:1237 (1987)]. Promoterand enhancer elements have been isolated from a variety of eukaryoticsources including genes in yeast, insect and mammalian cells and viruses(analogous control elements, i.e., promoters, are also found inprokaryotes). The selection of a particular promoter and enhancerdepends on what cell type is to be used to express the protein ofinterest. Some eukaryotic promoters and enhancers have a broad hostrange while others are functional in a limited subset of cell types (forreview, see, Voss et al. Trends Biochem. Sci., 11:287 (1986); andManiatis et al., supra (1987)). For example, the SV40 early geneenhancer is very active in a wide variety of cell types from manymammalian species and has been widely used for the expression ofproteins in mammalian cells [Dijkema et al., EMBO J 4:761 (1985)]. Twoother examples of promoter/enhancer elements active in a broad range ofmammalian cell types are those from the human elongation factor 1α gene[Uetsuki et al., J. Biol. Chem., 264:5791 (1989); Kim et al., Gene,91:217-223 (1990); and Mizushima and Nagata, Nuc. Acids. Res., 18:5322(1990)] and the long terminal repeats of the Rous sarcoma virus [Gormanet al., Proc. Natl. Acad. Sci. USA, 79:6777 (1982)] and the humancytomegalovirus [Boshart et al., Cell 41:521-530 (1985)].

As used herein, the term “promoter/enhancer” denotes a segment of DNAthat contains sequences capable of providing both promoter and enhancerfunctions (i.e., the functions provided by a promoter element and anenhancer element, see above for a discussion of these functions). Forexample, the long terminal repeats of retroviruses contain both promoterand enhancer functions. The enhancer/promoter may be “endogenous” or“exogenous” or “heterologous.” An “endogenous” enhancer/promoter is onethat is naturally linked with a given gene in the genome. An “exogenous”or “heterologous” enhancer/promoter is one that is placed injuxtaposition to a gene by means of genetic manipulation (i.e.,molecular biological techniques) such that transcription of that gene isdirected by the linked enhancer/promoter.

A regulatable promoter is one whose level of activity is subject toregulation by a regulatory molecule. An inducible promoter is one thatis normally substantially inactive, but that is activated by the bindingof an inducer to an operator site of the promoter. A repressiblepromoter is one that is normally active, but that is substantiallyinactivated by the binding of a repressor to an operator site of thepromoter. Similar terminology applies to enhancers.

The inducer or repressor molecules are typically expressed only inparticular tissues, at a particular developmental stage, or underparticular environmental conditions (e.g., damage to the cell,infection, overproduction of a metabolite, absence of a nutrient). Inthe absence of an inducer an inducible promoter may be inactive or mayproduce a low level of activity. The level of activity in the presenceof the inducer will be higher than the basal rate. A tightly induciblepromoter is one whose basal level of activity is very low, e.g., lessthan 10% of its maximum inducible activity.

Different promoters may have different levels of basal activity in thesame or different cell types. When two different promoters are comparedin a given cell type in the absence of any inducing factors, if onepromoter expresses at a higher level than the other it is said to have ahigher basal activity.

The activity of a promoter and/or enhancer is measured by detectingdirectly or indirectly the level of transcription from the element(s).Direct detection involves quantitating the level of the RNA transcriptsproduced from that promoter and/or enhancer. Indirect detection involvesquantitation of the level of a protein, often an enzyme, produced fromRNA transcribed from the promoter and/or enhancer. A commonly employedassay for promoter or enhancer activity utilizes the chloramphenicolacetyltransferase (CAT) gene. A promoter and/or enhancer is insertedupstream from the coding region for the CAT gene on a plasmid; theplasmid is introduced into a cell line. The levels of CAT enzyme aremeasured. The level of enzymatic activity is proportional to the amountof CAT RNA transcribed by the cell line. This CAT assay therefore allowsa comparison to be made of the relative strength of different promotersor enhancers in a given cell line. When a promoter is said to express at“high” or “low” levels in a cell line this refers to the level ofactivity relative to another promoter that is used as a reference orstandard of promoter activity.

Efficient expression of recombinant DNA sequences in eukaryotic cellsrequires expression of signals directing the efficient termination andpolyadenylation of the resulting transcript. Transcription terminationsignals are generally found downstream of the polyadenylation signal andare a few hundred nucleotides in length. The term “poly A site” or “polyA sequence” as used herein denotes a DNA sequence that directs both thetermination and polyadenylation of the nascent RNA transcript. Efficientpolyadenylation of the recombinant transcript is desirable astranscripts lacking a poly A tail are unstable and are rapidly degraded.The poly A signal utilized in an expression vector may be “heterologous”or “endogenous.” An endogenous poly A signal is one that is foundnaturally at the 3′ end of the coding region of a given gene in thegenome. A heterologous poly A signal is one that is one that is isolatedfrom one gene and placed 3′ of another gene. A commonly usedheterologous poly A signal is the SV40 poly A signal. The SV40 poly Asignal is contained on a 237 bp Bam HI/Bcl I restriction fragment anddirects both termination and polyadenylation [Sambrook et al., supra].

The cytomegalovirus immediate early promoter-enhancer (CMV-IE) is astrong enhancer/promoter. [See, Boshart et al., supra.] Another strongpromoter-enhancer for eukaryotic gene expression is the elongationfactor 1α promoter enhancer. [See, Kim et al., supra; and Mizushima andNagata, supra.]

The internal promoter for a transgene may be the promoter native to thattransgene, or a promoter native to the target cell (or viruses infectingthe target cell), or another promoter functional in the target cell.

The promoters and enhancers may be those exhibiting tissue or cell typespecificity that can direct the transgene expression in the target cellsat the right time(s). For example, a promoter to control humanpreproinsulin must be operable under control of carbohydrate in theliver. An example of such a promoter is the rat S-14 liver-specificpromoter.

Promoters (and enhancers) may be naturally occurring sequences, orfunctional mutants thereof, including chimeras of natural sequences andmutants thereof. For example, a tissue-specific, development-specific,or otherwise regulatable element of one promoter may be introduced intoanother promoter.

Chen et al, Proc. Nat. Acad Sci USA, 93:10057-10062 (1996) placed aVSV-G gene under the control of a tetracycline-inducible promoter andalso expressed a fusion of the ligand binding domain of the estrogenreceptor to a chimeric transcription factor, tTA, obtained by fusing thetet repressor (tetR) and the activation domain of HSV virion protein 16.

For the ability to replace the endogenous 5′ LTR promoters and enhancerswith heterologous ones, such as CMV immediate-early enhancer-promoter[see, Chang, et al., J. Virol., 67: 743-52 (1993)].

Vector; Transfection of Vectors

As used herein, the term “vector” is used in reference to nucleic acidmolecules that can be used to transfer nucleic acid (e.g., DNA)segment(s) from one cell to another. The term “vehicle” is sometimesused interchangeably with “vector.” It is intended that any form ofvehicle or vector be encompassed within this definition. For example,vectors include, but are not limited to viral particles, plasmids, andtransposons.

The term “transfection” as used herein refers to the introduction offoreign DNA into eukaryotic cells. Transfection may be accomplished by avariety of means known to the art including but not limited to calciumphosphate-DNA co-precipitation, DEAE-dextran-mediated transfection,polybrene-mediated transfection, electroporation, microinjection,liposome fusion, lipofection, protoplast fusion, retroviral infection,and biolistics.

Vectors may contain “viral replicons” or “viral origins of replication.”Viral replicons are viral DNA sequences that allow for theextrachromosomal replication of a vector in a host cell expressing theappropriate replication factors. Vectors that contain either the SV40 orpolyoma virus origin of replication replicate to high copy number (up to10⁴ copies/cell) in cells that express the appropriate viral T antigen.Vectors containing the replicons from bovine papillomavirus orEpstein-Barr virus replicate extrachromosomally at low copy number(about 100 copies/cell).

Expression Vector

The term “expression vector” as used herein refers to a recombinant DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host organism. Nucleic acid sequencesnecessary for expression in prokaryotes usually include a promoter, anoperator (optional), and a ribosome binding site, often along with othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals. In some embodiments,“expression vectors” are used in order to permit pseudotyping of theviral envelope proteins.

Host Cells

The host cell is a cell into which a vector of interest may beintroduced and wherein it may be replicated, and, in the case of anexpression vector, in which one or more vector-based genes may beexpressed.

It is not necessary that the host cell be injectable by the transgenevector virions of the present invention. Indeed, in some examples theynot be so infectable, so the host cells do not bind the virions andthereby reduce the vector production titer. This can be achieved bychoosing (or engineering) cells that do not functionally express thereceptor to the vector particle envelope protein.

Target Cells and Organisms

The transgene vector may be administered to a target organism by anyroute that will permit it to reach the target cells. Such route may be,e.g., intravenous, intratracheal, intracerebral, intramuscular,subcutaneous, or, with an enteric coating, oral. Alternatively, targetcells may be removed from the organism, infected, and they (or theirprogeny) returned to the organism. Or the transgene vector may simply beadministered to target cells in culture.

The target cells into which the transgene is transferred may be any cellthat the transgene vector, after packaging into a virion, is capable ofinfecting, and in which the control sequences governing expression ofthe transgene are functional. Generally speaking, it will be aeukaryotic cell, such as a vertebrate cell (e.g., a cell of a mammal orbird). If a mammal, the mammal may belong to one of the ordersArtiodactyla (e.g., cows, pigs, goats, sheep), Perissodactyla (e.g.,horses), Rodenta (e.g., rats, mice), Lagomorpha (e.g., rabbits),Carnivora (e.g., dogs, cats) or Primata (e.g., humans, apes, monkeys,lemurs). If a bird, it may be of the orders Anseriformes (e.g., ducks,geese, swans) or Galliformes (e.g., quails, grouse, pheasants, turkeys,chickens). In one embodiment, it will be a human cell. The cells inquestion may be dividing or non-dividing cells. Non-dividing cells ofparticular interest include airway epithelial cells, nervous system(e.g., central nervous system) cells, or hepatocyte cells. Examples ofnervous system cells include, without limitation, neurons, glia (e.g.,astrocytes), and progenitor cells.

Dividing cells of particular interest include hematopoietic stem cells,muscle cells, white blood cells, spleen cells, liver cells, epithelialcells, and eye cells.

TE671, HepG2, HeLa, 293T, and MT4 are of particular interest forexperimental studies. TE671 rhabdomyosarcoma cells can be induced todifferentiate into muscle cells by HIV-1 Vpr. HepG2 hepatoma, HeLacervical carcinoma, 293T human kidney carcinoma and MT4 lymphoma cellsare all transformed by HTLV-I human T cell leukemia virus type I. MT4cells are very susceptible to wild-type HIV-1 NL4-3 and hence have beenused as indicator cell for RCV.

Miscellaneous Definitions

As used herein, the term “endogenous virus” is used in reference to aninactive virus that is integrated into the chromosome of its host cell(often in multiple copies), and can thereby exhibit verticaltransmission. Endogenous viruses can spontaneously express themselvesand may result in malignancies.

The term “gene” refers to a DNA sequence of a vector or genome thatcomprises a coding sequence and is operably linked to one or morecontrol sequences such that, in a suitable host cell, under suitableconditions, a biologically active gene product, or a gene product thatis a precursor of a biologically active molecule, is produced that isencoded by the coding sequence. This gene product may be atranscriptional product, i.e., a messenger RNA, as in the case of anantisense RNA or a ribozyme. Or it may be a translational product, i.e.,a polypeptide (the term “polypeptide” as used herein includesoligopeptides), which is either biologically active in its own right, orfurther processed by the cell to generate one or more biologicallyactive polypeptide products. In the case of retroviruses, where thegenome is RNA, the term “gene” also refers to the RNA sequence of theretroviral genome that a suitable host cell reverse transcribes into aDNA sequence that acts as a gene in the classic sense.

Depending on context, the term “gene” may refer to the DNA sequenceencoding a single mRNA transcript, or only to that portion of the DNAsequence that is ultimately expressed as a single polypeptide chain.

In the vectors of the present invention, each gene may be constructedfrom genomic DNA, complementary DNA (DNA reverse transcribed from mRNA),synthetic DNA, or a combination thereof. The gene may duplicate a genethat exists in nature, or differ from it through the omission of introns(noncoding intervening sequences), a so-called mini-gene, silentmutations (i.e., mutations that do not alter the amino acid sequence ofthe encoded polypeptide), or translated mutations (i.e., mutations thatdo alter that sequence). In the latter case, the mutations may befunctional mutations (ones that preserve at least a substantial portionof at least one of the biological activities or functions of the encodedpolypeptide) or nonfunctional (inactivating) mutations.

As used herein, the term “transcription unit” refers to the segment ofDNA between the sites of initiation and termination of transcription andthe regulatory elements necessary for the efficient initiation andtermination. For example, a segment of DNA comprising anenhancer/promoter, a coding region and a termination and polyadenylationsequence comprises a transcription unit.

Assays

From time to time, one may wish to ascertain various informationconcerning the envelope, packaging and transgene vectors of the presentinvention.

One might like to know whether the vectors have become established inthe cell; whether particular vector genes have integrated into thegenome; whether the packaging cell line is producing viral proteins;whether those viral proteins are being assembled into viral particles;whether, in the absence of the transgene vector, those viral particlesare essentially free of RNA, such as packaging vector RNA; whetherrecombination occurs between the packaging vector and the transgenevector, or between these two vectors and defective retrovirusesendogenous to the host (or target) cell; whether such recombination, ifany, produces replication-competent virus; whether recombinant virus ispackaged by the packaging cell line; the efficiency with which thepackaging cell line packages the transgene vector into the viralparticles; whether the transgene vector-containing viral particles areinfectious vis-a-vis the target cells; whether the latter particles arecytotoxic to the target cells; whether the latter particles areimmunogenic to the target organism; whether infected target cellsthemselves produce viral RNA-containing particles, infectious orotherwise; and the level and duration of expression of the transgene inthe target cells.

The successful establishment of the envelope, packaging or transgenevector in the host (or target) cell may be verified by selecting for thepresence of a selectable marker, or screening for the presence of ascreenable marker, carried by the vector. The integration of therelevant envelope, packaging or transgene vector genes may be determinedby collecting genomic DNA, amplifying the gene of interest by PCR, anddetecting the amplified sequence with a suitable hybridization probe.The production of viral proteins may be detected by an immunoassay; thesample may be a cell lysate or a cell supernatant. An immunoassay byitself cannot determine whether the viral proteins are produced infunctional form, although there is greater assurance of this if theantibody used is directed to a conformational epitope, or is anactivity-neutralizing antibody. One may alternatively detect theappropriate messenger RNA by means of a hybridization probe.

The functionality of the produced Gag and Env protein may be determinedby examining the cell lysate or supernatant for the presence of viralparticles; these may further be examined for proper morphology by meansof an electron microscope. It is also possible that antibodies could beused that bind to the formed viral particles, but not to gp120 or gp41by itself. The functionality of the Pol reverse transcriptase may bedetermined by assaying the viral particles for RT activity. Thefunctionality of the Pol integrase is apparent only in assays thatexamine whether RNA from viral particles is integrated into the targetcell.

Viral particles produced by the packaging cell line may be collected andassayed for total RNA content. If more specific information is desiredas to the nature of any packaged RNA, a suitable hybridization probe maybe employed.

In an infectivity assay, the vector is introduced into a first cultureof susceptible cells. Then, either a second culture is layered onto thefirst, so that infectious particles may travel by cell-to-cell contact,or the second culture is exposed to the supernatant of the firstculture. The cells of the first and second culture are examined for aleast one of the following indicia: RT activity, p24 Gag antigenexpression, production of viral particles, and cytotoxic effects. Thestringency of the assay is dependent on the susceptible of the cells toinfection and to cytotoxicity, and the time allowed for therecombination and spread of the virus in the first and second cultures.Typically, the infectivity of the vector or vector system will becompared with that of a wild-type, unattenuated, replication-competentretrovirus.

Animal studies may be used to ascertain the immunogenicity andpathogenicity of the vector system.

Measurement of Infectivity of Packaging Vector Per Se

The ability of a packaging vector to generate transmissible virus, asopposed to defective virus, may be measured. One method is described byMann et al., Cell, 33:153-159 (1983). The packaging vector and itswild-type counterpart are independently transfected into suitable hostcells, and reverse transcriptase activity in the culture supernatants isassayed over a period of days or weeks. A rapid increase in RT activityover 24-48 hrs is indicate of gene expression after transienttransfection. A continued increase is indicative of the efficient spreadof virus from the initially transfected cells to the remaining cells onthe plate.

A slow or delayed increase could be indicative of either a steady butattenuated spread of virus, or to generation of competent virus bymutation, or by recombination with a cellular sequence capable ofproviding the missing function. To differentiate these possibilities,one may use various dilutions of culture supernatants from cellspreviously transfected (days or weeks before) with the vector (or withthe control virus), use them to infect fresh cells, and monitor RTactivity in the latter. If the latter cells develop high levels of RTactivity, it suggests that non-defective virus was present in thetransferred culture supernatant.

Measurement of Packaging Efficiency

The packaging efficiency of a packaging cell line in the presence orabsence of the packageable transducing transgene vector may be measuredin a variety of ways (see, e.g., Mann et al., supra). In essence, totalcellular RNA is purified from the culture supernatant of the test andcontrol cell lines, and viral RNA is extracted from purified viralparticles released from the test and control cell lines. The two virionpreparations are normalized by reference to their reverse transcriptaseactivity just prior to RNA extraction. The purified RNAs are probed witha virus-specific hybridization probe (e.g., a plasmid containing theentire viral genome) in a slot-blot assay, and the amount of viral RNAin the particles and in the cells is thereby quantified.

It is not unusual for the packaging efficiency of a packaging cell lineto be less than 1% that of a host cell infected by wild-type virus.

Measurement of Packaging Specificity

It is also desirable that the packaging cell line be able to efficientlypackage the highly defective transgene vector into viral particles, andbud the particles into the culture supernatant (in vitro) orextracellular environment (in vivo) without also budding helper virus(the packaging vectors).

One method of measuring this packaging specificity is described by Mannet al. (supra). In essence, the transgene vector is transfected into thepackaging (helper) cell line. After 24 hours, the culture supenatantsare used to infect fresh potential host cells (reporter cells). Two dayslater, selection pressure for the transferred gene is applied, and 8-10days later, the transferred gene-positive colonies or cells are counted.In addition, one determines the reverse transcriptase activity of thesupernatant collected from the packaging cell lines, and the reversetranscriptase activity of the fresh cells. A transgene vector-specificpackaging cell line will produce a high transfer gene activity and a lowreverse transcriptase activity in the reporter cells. In addition, thereporter cells will not produce reporter gene-positive colony-formingunits (cfus).

Measurement of Helper Activity

The ability of a packaging vector to provide all viral functionsrequired in trans may be assayed by co-transfecting host cells with thepackaging vector (or control virus) and with a reporter vector carryinga selectable reporter gene. After 24 hours, culture supernatants of thetransfected cells are used to infect a second plate of host cells.Selection pressure for the reporter gene is applied, andreporter-positive colonies are counted. If the helper activity is ofwild-type magnitude, the count for the packaging vector should be of thesame order of magnitude as that for the control virus, and no reporteractivity should be detectable in the second plate when the reportervector or the control wild-type virus expressing all viral functions istransfected into the host cells of the first plate by itself.

Measurement of Generation of Replication-Competent Virus (RCV)

Several sensitive assays are available for the detection of RCV in thepresent retroviral vector systems. These include: (1) co-cultivationwith a sensitive cell line such as MT4, AA2 or PBLs; (2) the CD4 HeLaMAGI cell assay that relies on Tat transactivation of an integratedLTR-lacZ gene; and (3) a sensitive immunohistochemical staining methodfor the detection of HIV antigen expression at the individual celllevel.

RC-HIV can also be studied in an in vivo model by transduction ofhumanized SCID/beige mice. In the latter model, a long in vivoincubation time can be performed, mimicking the situation that exists ina human clinical trial. In addition, the possibility of generatingHIV/HERV recombinants may be carefully tested using an artificiallyconstructed HIV/HERV-env recombinant.

Virion Stability

Since one class of the therapeutic agents of the present invention wouldbe the packaged transgene vectors, the stability of the packagedtransgene vectors under adverse conditions, especially those that mightbe encountered during storage, is of interest. Thermostability may beascertained by subjected them to elevated (e.g., 37° C.) or depressed(e.g., 4° C.) temperatures for various periods of time (e.g., 2, 4, 6 or8 hrs., or overnight), or to a number (e.g., 1-6) freeze-thaw cycles,and determining the number of infectious particles remaining as apercentage of the number of such particles prior to treatment.

Assays for Immunogenicity

A method for determining whether the contemplated vectors, or their geneproducts, could elicit an immune response in a subject involvesevaluating cell-mediated immunity (CMI) using either an immunocompetentmouse model or a humanized SCID/beige mouse model.

Using a modified hu-PBL-SCID mouse reconstitution protocol, an in vivomodel for evaluating CMI against HIV-1 in humans has been developed.SCID/beige mice lacking T, B and natural killer (NK) cell functions areseverely immunodeficient. This strain of mice can be successfullyreconstituted with fresh human peripheral blood lymphocytes (PBLs), andexhibits functional human naive, memory and activated T cell markers formore than 2-3 months. In these experiments, spleen and peripheral bloodlymphocytes were harvested 38 days after reconstitution fromreconstituted SCID/beige mice, and red blood cells were lysed prior toincubation with anti-mouse 2 Kd, anti-human CD45, anti-human CD3,anti-human CD4 and anti-human CD8 labeled antibodies. Reconstitutedhuman lymphoid cell populations in the spleen and in the peripheralblood of the SCID/beige mice can reach up to 50-80% and 5-12%,respectively.

For the immune response study, mice repetitively injected with the viralvectors will be analyzed. Their sera will be assayed for Ab response toviral antigens, such as p24 Gag or the pseudotype env. For cell-mediatedimmune response study, the mouse splenocytes will be isolated and an invitro assay for cellular immunity will be performed as described below.T cell response to recall antigen is normally characterized by theproduction of interferon gamma (IFNγ). This assay requires activation oflymphocytes with the test Ags, such as Gag p24 or Gag-Pol or envproteins of the vector.

Upon activation, the Th1 lineage of T cells produce interferon gamma(IFN-γ) and the measurement of IFN-γ production has been shown to be areliable assay for CMI. Thus, for example, to determine CMI againstHIV-1 using the in vivo humanized SCID/beige mouse model, a sensitiveELISPOT assay for the detection of IFN-γ producing cells was developed.With the computer assisted imaging system integrated into this protocol,the ELISPOT method was shown to be very convenient and more sensitivethan the conventional limiting dilution assay for the determination ofthe effector T cell precursor frequency. This in vivo model and theELISPOT assay system were developed for the evaluation of in vivo CMIafter lentiviral gene transfer. See, e.g., PCT/US98/06944.

Pseudotyping Retroviral Vectors with Novel Envelope Glycoproteins toEnhance Target Cell Transduction.

Retroviral vector-mediated gene transfer begins with the attachment ofthe virion to a specific cell surface receptor [Goff, J. Gene Med.3:517-528 (2001)]. This attachment is the first step in the entire genetransfer process and a crucial factor in determining vector tropism andthe range of target tissues/cell types. Vector binding is mediated byspecific interactions between the envelope glycoproteins on the virionand one or more surface receptor molecules on the target cell. If thisreceptor molecule is absent (as when its expression is specific forcertain cell types) or is variant in the binding region (such as inspecies other than the natural host), gene transfer cannot occur [Coffinet al. Retroviruses. Plainview: Cold Spring Harbor Press, 2000]. Byreplacing the native envelope protein with other retroviral ornon-retroviral glycoproteins, a process termed “pseudotyping”, one canalter the host range of the vectors, which can result in increasedtransduction efficiency of desirable target cells [Miller, Proc. Natl.Acad. Sci. USA, 93:11407-11413 (1996)].

The vesicular stomatitis virus (VSV-G) and the amphotropic envelopeproteins are the two most commonly used glycoproteins forretroviral/lentiviral-based gene transfer. However, for many cell typesthe transduction efficiency using both envelope glycoproteins is low.Moreover, both envelopes have limitations for potential clinicalapplications. For example, VSV-G is cytotoxic [Park et al., Blood96:1173-1176 (2000); Stewart et al., Proc. Natl. Acad. Sci. USA96:12039-12043 (1999)] and may be inactivated by human serum [DePolo etal., Mol. Ther., 2:218-222 (2000)] and the amphotropic glycoprotein isfragile and does not tolerate centrifugation concentration as well asVSV-G [Burns et al. Proc. Natl. Acad. Sci. USA, 90:8033-8037 (1993)]. Inan effort to increase the in vivo gene transfer efficiency of lentiviralvectors to cells and tissues for therapeutic gene delivery, the FIVvector was pseudotyped with viral envelope glycoproteins fromnon-retroviral enveloped viruses. The gene transfer efficiency of thesepseudotyped vectors were evaluated in several cells and tissuesincluding airway epithelia, lung, brain, and liver.

Pseudotyping Retroviral Vectors with LCMV Envelope Glycoproteins.

The present invention provides a method to pseudotype retroviruses toattain high titers suitable for ex vivo and in vivo gene transfer usingenvelope glycoproteins from lymphocytic choriomeningitis virus (LCMV)strain WE54. This invention is useful for gene therapy applicationsusing retroviral vectors. The methods described in this disclosureprovide a novel way to increase the transduction efficiency and targetspecific cell types that were previously poorly accessible with existingvector envelopes. In particular these methods have applications fortargeting tissues such as airway epithelia, neural progenitors in theCNS, hepatocytes and others.

Further methods described herein allow for modification of specificamino acid residues of the glycoproteins to further enhance transductionefficiency. The methods facilitates the production of stable packagingcell lines for vector production.

The invention disclosed has applications to the field of gene therapy.The approaches described overcome previous limitations efficientlytransducing cells with retroviral vectors using glycoproteins fromenveloped viruses. The LCMV-WE54 pseudotyped vector has applications fordiseases affecting airway epithelia (i.e., cystic fibrosis), the CNS(i.e., neurodegenerative or neurogenetic disorders), and hepatocytes(i.e., hemophilia).

The inventors initially documented that the LCMV envelope from WE54strain (also called WE-HPI) was compatible with production of thenon-primate lentiviral vector feline immunodeficiency virus based vector(FIV). Titers were sufficiently high to permit in vitro and in vivostudies. The inventors documented that the LCMV-WE54 pseudotyped vectortransduced human airway epithelia much more efficiently from the apicalrather than the basolateral surface in vitro. Subsequently, a singlepoint mutation (L260F) was made in the glycoprotein. Vector preparationswith this modified LCMV-WE54 envelope transduced airway epithelia with˜10-fold greater efficiency than the wild type envelope. The efficiencyof gene transfer with the L260F LCMV-WE54 envelope is greater than thatobtained with several other conventional envelopes.

Additional mutants including S153F and the double mutant L260F/S153F aremade. These changes are made to reduce the affinity of the vector to oneidentified receptor called alpha-dystroglycan.

As described in the Examples below, experiments in cultured human airwayepithelia, mouse liver, and mouse brain document that the vector isfunctional for in vivo gene transfer. Additional experiments showedtransduction of cells in olfactory bulb (OB) brain cells followingintrastriatal injection in mice, and indicated that the vectortransduced neural progenitor cells that migrated from the striatum tothe OB. Intravenous injection of the vector in the mouse resulted insignificant transduction of the liver, including hepatocytes.

Methods of Preparing Retroviral Vectors

Recombinant retroviruses can be produced by a number of methods. Onemethod is the use of packaging cell lines. The packaging cells areprovided with viral protein-coding sequences, such as encoded on twothree plasmids [Johnston et al., J. Virol., 73:4991-5000 (1999)]. Theplasmids encode all proteins necessary for the production of viableretroviral particles and encode a RNA viral construct that carries thedesired gene (e.g., the gene encoding the mutant envelope protein or amutant envelope fusion protein), along with a packaging signal (Ψ Psi)that directs packaging of the RNA into the retroviral particles.

Alternatively, the mutated retroviral genome can be transfected intocells using commonly known transfection methods such as calciumchloride, electroporation, or methods described in the examples. [Seealso, Sambrook et al. Molecular Cloning: A Laboratory Manual (2^(nd)ed.), Cold Spring Harbor Laboratory Press (1989).]

The retroviral vector may also include an appropriate selectable marker.Examples of selectable markers that may be utilized in either eukaryoticor prokaryotic cells, include but are not limited to, the neomycinresistance marker (neo), the ampicillin resistance marker (amp), thehygromycin resistance marker (hygro), the multidrug resistance (mdr)gene, the dihydrofolate reductase (dhfr) gene, the beta-galactosidasegene (lacZ), and the chloramphenicol acetyl transferase (CAT) gene.

Cells transfected with cDNAs encoding a retrovirus genome or infectedwith retrovirus particles can be cultured to produce virions or virusparticles. Virus particle-containing supernatant can be collected. Thevirus particles can be concentrated by centrifuging the supernatant,pelleting the virus and by size exclusion chromatography. Pharmaceuticalcompositions containing virus particles can also be resuspended inpharmaceutically acceptable liquids or carriers such as saline.

Retroviral Gene Transfer

The retrovirus particles described above can infect cells by the normalinfection pathway as along as recognition of the target cell receptor,fusion and penetration into the cell all occur. All eukaryotic cells arecontemplated for infection by the recombinant virions. For example, thecells used in the present invention can include cells from vertebrates(e.g., human cells).

The vectors of the present invention can be used in vivo with a numberof different tissue types. Examples include airway epithelia, liver, andneural (e.g., central nervous system) cells. Methods for infecting cellswith retrovirus particles are described generally in Gene TherapyProtocols: Methods In Molecular Medicine, Paul D. Robbins (ed.) (HumanaPress, 1997). Other methods of preparing and administering retroviralparticles in gene therapy commonly known to the skilled artisan may beused.

The types of genes that are to be transferred into the host cell by theretrovirus particles of this invention may encode therapeutic agents,enzymes, growth factors, cell receptors, suicide or lethal genes.

Such genes or nucleic acid molecules are under the control of a suitablepromoter. Suitable promoters, which may be employed, include, but arenot limited to adenoviral promoters, the cytomegalovirus promoter, theRous sarcoma virus (RSV) promoter, the respiratory syncytial viruspromoter, inducible promoters such as the metallothionein promoter, heatshock promoters, or the gene's own natural promoter. It is to beunderstood however, that the scope of the present invention is not to belimited to specific foreign genes or promoters.

Most gene therapy is administered to cells ex vivo. The cells receivingsuch gene therapy treatment may be exposed to the retrovirus particlesin combination with a pharmaceutically acceptable carrier suitable foradministration to a patient. The carrier may be a liquid carrier (forexample, a saline solution), or a solid carrier such as an implant ormicrocarrier beads. In employing a liquid carrier, the cells may beintroduced intravenously, subcutaneously, intramuscularly,intraperitoneally, intralesionally, etc. In yet another embodiment, thecells may be administered by transplanting or grafting the cells. Lipiddestabilizers, such as thiocationic lipids, can be utilized in admixturewith the viral vector or liposomal vector to increase infectivity (seeexamples of lipid destabilizers in U.S. Pat. Nos. 5,739,271 and5,711,964).

Although most current gene therapy protocols involve ex vivotransfection of cells, the vectors disclosed would permit in vivotreatment of a subject, such as a human patient, as well as ex vivoutilization. For example, ex vivo therapy requires that cells such ashepatocytes be removed from the patient, transduced with the retroviralparticle containing the desired nucleic acid molecule, and thentransplanted back into the patient. In vivo therapy would allow directinfusion of the gene therapy vector, without the intervening steps andthe complications that they raise. Moreover, this will allow access totissues that may not have been good candidates for ex vivo gene therapy.

Virus Particles

Gene therapy vectors also include pseudotyped virus particles.Pseudotype viruses were originally created to overcome problemsencountered by gene therapy vectors' natural host cell tropisms. Inrecent years, many gene therapy patents have issued wherein the vectorcontains a heterologous polypeptide used to target the vector tospecific cells, such as vectors containing chimeric fusion glycoproteins(U.S. Pat. No. 5,643,756); vectors that contain an antibody to a viruscoat protein (U.S. Pat. No. 5,693,509); viruses engineered to allowstudy of HIV-1 in monkeys, a species that normally cannot be infected byHIV-1, by creating hybrid viruses (U.S. Pat. No. 5,654,195); andpseudotype retrovirus vectors that contain the G protein of VesicularStomatitis Virus (VSV) (U.S. Pat. Nos. 5,512,421 and 5,670,354). In thecurrent invention an exemplary envelope protein is baculovirus GP64, andrelated envelopes.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 LCMV Pseudotyped FIV Targets the Apical Surface ofAirway Epithelia for Entry

The inventors evaluated the ability of LCMV glycoproteins (GPs) topseudotype the FIV vector. LCMV-GP is initially expressed as a precursorpolypeptide, GP-C, which is post-translationally processed by a cellularprotease into GP1 and GP2. GP1 meditates binding to the cellularreceptor for LCMV, and GP2 contains the fusion peptide and thetransmembrane region [Buchmeier et al., supra]. Recently Beyer andcolleagues reported successful pseudotyping of HIV-based lentivirusvectors with glycoproteins from the LCMV WE54 strain [Beyer et al., J.Virol. 76:1488-1495 (2002)]. In contrast to amphotropic-MLV vectorparticles, LCMV pseudotypes could be efficiently concentrated byultracentrifugation without loss of vector titer. The inventors obtainedthe LCMV strain WE54 GP from Dr. Beyer and evaluated its efficiency inpseudotyping FIV. Titers for the FIV pseudotyped with LCMV WE54 were˜5×10⁸ TU/ml, very suitable for in vitro and in vivo studies.

High titer FIV pseudotyped with the LCMV-WE54 GPs was evaluated for itsability to transduce primary cultures of well-differentiated humanairway epithelia. The vector was applied to the apical or basolateralsurface of the epithelial sheet at an MOI of ˜5. For comparison,VSVG-FIV results from previous studies are shown [Sinn et al., J. Virol.77:5902-5910 (2003)]. Four days later, gene transfer was quantified bymeasuring beta-galactosidase activity as described previously [Sinn etal., supra]. As shown in FIG. 1, while the LCMV pseudotyped vectortransduced the cells from either surface, the apical efficiency wassignificantly greater than basolateral. This finding is the opposite ofthe inventors' findings with the VSV-G envelope, where entry ispredominantly basolateral [Sinn et al., supra; Wang et al., J. Clin.Invest. 104:R49-R56 (1999)]. It is important to note that at the sameMOIs, the beta-galactosidase enzyme activity levels for the LCMV vectorwere similar to those achieved with the VSV-G envelope, the mostefficient pseudotype we have identified to date. Gene transfer wasinhibited by the reverse transcriptase inhibitor AZT, indicating thatthese results do not represent protein transfer or pseudotransduction.

Arenaviruses using alpha-DG as their major receptor have the smallaliphatic amino acids leucine or isoleucine at position 260 in GP1 and abulky aromatic amino acid, phenylalanine or tyrosine, at position 259 ofGP1 [Spiropoulou et al., J Virol., 76:5140-5146 (2002)]. While thereceptor for the LCMV-WE54 strain the inventors used to pseudotype FIVhas not been identified in airway epithelia, the LCMV-WE54 strain has anF at position 259 and an L at position 260, and is predicted to bindalpha-DG with high affinity. The inventors hypothesized that FIVpseudotyped with wild type LCMV-WE54 GPs uses alpha-DG as a highaffinity receptor for initial binding steps in polarized human airwayepithelia. The inventors mutated the L at position 260 in the LCMV-WE54envelope to an F (L260F) [Spiropoulou et al., supra] to test itsimportance in directing LCMV WE54 transduction of differentiated humanairway epithelia. Both WE54 and the L260F mutants pseudotype FIV withcomparable high titers. FIG. 2 shows the gene transfer efficiency of FIVpseudotyped with these two envelopes following apical or basolateralapplication to well-differentiated primary cultures of human airwayepithelia. The L260F variant transduces the cells much more efficientlythan WE54 and maintains an apical preference for entry.

Example 2 Receptors for LCMV

Alpha-dystroglycan (alpha-DG) was identified as a high affinity receptorfor several arenaviruses including some strains of LCMV, Lassa virus,Oliveros virus, and Latino virus [Cao et al., Science, 282:2079-2081(1998)]. Dystroglycan is a dystrophin-associated glycoprotein thatconnects the cytoskeleton with the extracellular matrix and is widelyexpressed in most tissues, including the lung [Henry and Campbell, Cell,95:859-870 (1998); White et al., Am. J. Respir. Cell. Mol. Biol.24:179-86 (2001)]. Dystroglycan is post-translationally cleaved into ahighly glycosylated peripheral membrane protein alpha-DG, which isnoncovalently associated with the membrane-spanning proteinbeta-dystroglycan [Henry and Campbell, Curr. Opin. Cell Biol.,11:602-607 (1999)]. Beta-dystroglycan is linked to the cytoskeleton. Itis important to note that studies with wild type LCMV WE54 straindemonstrate high affinity binding to alpha-DG (Spiropoulou et al.,supra). However, some LCMV variants (e.g., Armstrong strain)inefficiently interact with alpha-dystroglycan and also infectdystroglycan-negative cells. Therefore, there appears to be at least oneadditional, currently unknown receptor or co-receptor for LCMV[Spiropoulou et al., supra].

To investigate the potential role for alpha-dystroglycan as a receptorfor LCMV pseudotyped FIV, the inventors evaluated the ability oflaminin, IIH6 antibody, or cell culture media containing shed alphadystroglycan to inhibit gene transfer to airway epithelia with the WE54or L260F pseudotypes. Work of others indicated that the WE54 GP1 bindsalpha DG with high affinity. Cells were treated with the respectivereagents for one hour prior to application of vector (MOI=1) andthroughout the four hour application period. As shown in FIG. 3, each ofthese reagents had a small inhibitory effect on WE54, with the greatestblocking effect coming from the cell culture media. None of the agentsaffected gene transfer with the L260F pseudotype. The findings supportthe notion that wild type WE54 has affinity for alpha DG while the L260Fpseudotype has little or no affinity for alpha DG. Thus, while theFIV-WE54 LCMV pseudotype may bind to alpha DG with some affinity, thereis most likely another receptor or co-receptor involved in thetransduction of airway epithelia. Furthermore, alpha DG does not appearto be an important receptor for the L260F variant.

Example 3 Gene Transfer to Mouse Liver Using FIV Pseudotyped with LCMVEnvelope

Gene transfer to the liver has potential clinical applications for manydiseases. The inventors evaluated the liver transduction properties ofthe FIV pseudotyped with LCMV-L260F. For systemic vector delivery to theliver, C57BL/6 mice received the LCMV pseudotyped FIV vectorintravenously via the tail vein using methods as previously described[Stein et al., Mol. Ther., 3:850-856 (2001); Kang et al., J Virol.,76:9378-9388 (2002)]. Three weeks later the animals were killed and theliver tissues examined for beta-galactosidase expression. Widespreadexpression was observed throughout the liver samples. This tropism forliver is useful for the production of secreted proteins or treatment ofdisorders primarily involving liver parenchyma, such as themucopolysaccharidoses.

Example 4 Targeting Cells in the CNS with LCMV Pseudotyped FIV

The inventors evaluated the gene transfer properties of FIV pseudotypedwith the LCMV-WE54 strain GP in the murine CNS. The plasmidLCMVgp.FIVθgal was introduced into the brain by striatal injection, andtissue samples were assessed for beta-gal labeling and staining withanti-NeuN antibodies, which served as markers for neurons. Theseexperiments revealed that FIV pseudotyped with the envelope from LCMVstrain WE54 (identical to WE-HPI) directs transgene expression in the OBafter striatal injection, suggesting that neural progenitor cells in thesubventricular zone (SVZ) were targeted. In support of this, transducedcells in the SVZ and the RMS also were noted, characteristic ofprogenitor cells and their progeny [Doetsch F, et al., Proc. Natl. Acad.Sci. USA, 96(20):11619-11624 (1999); Doetsch et al., Cell, 97(6):703-716(1999); Alvarez-Buylla et al., Brain Res. Bull., 57(6):751-758 (2002)].There were no co-labeled beta-gal/NeuN⁺ cells in the RMS or the SVZ.These very intriguing and exciting results indicated that pseudotypedLCMV vectors can be used to direct secretion of recombinant enzymes orgrowth factors from cells migrating along the rostral migratory streamfor a beneficial effect. The results also suggest that such a strategycan be used to effect a substantial correction and functional recoveryof CNS deficits in neurodegenerative or neurogenetic disorders such aslysosomal storage diseases (e.g., leukodystrophies,mucopolysaccharidoses, and ceroid lipofuscinoses), Huntington's disease,Parkinson's disease, amyotrophic lateral sclerosis, ataxias,dentatorubral-pallidoluysian atrophy, prion disease, and Alzheimer'sdisease. The method can also be used to transduce cells that are capableof dividing within the brain.

TABLE 1 Glycoproteins used for FIV brain gene transfer: predominant celltype transduced. VSV-G gp64 RRV LCMV neurons neurons, glia progenitorcells neuroglia, (SVZ), neurons (OB) ependyma, CP CP, choroid plexusSVZ, subventricular zone OB, olfactory bulb

Example 5 Targeting Neural Progenitor Cells with LCMV Pseudotyped FIV

Plasmids and vector production: In this study, fourreplication-incompetent FIV vectors were generated.VSV-G/FIV-CMVntbeta-gal is pseudotyped with the VSV-G envelope, and theCMV promoter drives expression of ntbeta-gal. LCMV/FIV-CMVntbeta-gal isLCMV pseudotyped and the CMV promoter drives expression of ntbeta-gal.LCMV/FIV-CMVbeta-gal is the same as the previous vector except that thebeta-gal is cytoplasmic; and LCMV/FIV-GFAPcre is LCMV-pseudotyped andthe GFAP promoter drives expression of cre recombinase.

FIV vectors were generated essentially as described [Stein and Davidson,Meth. Enzymol., 346:433-454 (2002)] by triple transfection of 293T cellswith the gag-pol-rev packaging plasmid, the env plasmid, and the vectorplasmid. The parental plasmid used to construct the vector plasmids waspVET_(L)Cmcs, which contains a multiple cloning site (mcs) downstream ofthe CMV promoter. pVET_(L)Cmcs was derived from pVET_(L)Cθ [Johnston etal., J Virol., 73:4991-5000 (1999)] by replacing the lacz gene (θ) withthe mcs. Coding regions for ntbeta-gal and beta-gal were subcloned intothe mcs of pVET_(L)Cmcs to generate the pFIVCMVntbeta-gal andpFIVCMVbeta-gal vector plasmids, respectively. To generate thepFIVGFAPcre vector plasmid, modifications were first made toPVET_(L)Cmcs. The major splice donor site was mutated from GT to AT, andthe RRE was moved from its native 3′ location to a site just upstream ofthe CMV, to generate pFIVdSDrreC. The CMV promoter was removed frompFIVdSDrreC and replaced with the human GFAP promoter sequence togenerate pFIVGFAP. The source of the GFAP promoter, pGfa2LAC-1, waskindly provided by Michael Brenner (University of Alabama, Birmingham,Ala.). This plasmid contains sequences −2163 to +47 of the human GFAPgene, in which the ATG has been mutated to TTG at position +15 (GenBankAccession No. M67446) [Besnard et al., J. Biol. Chem., 266:18877-18883(1991)]. The Bacteriophage P1 cre recombinase coding region (GenBankAccession No. X03453) with a PCR-primer generated Kozak sequence(ACCATG) was digested from pVET_(L)CMVcre [Sinnayah et al. Physiol.Genomics, 18:25-32 (2004)], and inserted into pFIVGFAP, immediatelydownstream of the GFAP promoter to generate pFIVGFAPcre. The env plasmidencoding the LCMV envelope glycoprotein of the WE54 strain, which is thesame as WE-HPI (GenBank Accession No. AJ318512) [Beyer et al., J Virol.,76:1488-1495 (2002)], was kindly provided by Winfried R. Beyer(Heinrich-Pette Institute, Hamburg, Germany). All vectors wereconcentrated from culture supernatants by centrifugation. Visual titersfor beta-gal and ntbeta-gal vectors were determined as described (Steinand Davidson, supra) by limiting-dilution transduction of HT1080 cellsand X-gal staining, and are expressed as transducing units/ml (TU/ml).Real-time PCR titers (integrating Units/ml, IntU/ml) for vectors weredetermined by isolation of genomic DNA from transduced HT1080 cells(Wizard kit, Promega Corp, Madison, Wis.) and quantifying integratedvector sequences.

Animals and Injections: C56BL6/J mice were purchased from The JacksonLaboratory (Bar Harbor, Me.) or were bred in-house. ROSA26 Cre reportermice [Soriano, Nat. Genet., 21:70-71 (1999)], on a C57BL6 backgroundwere purchased from The Jackson Laboratory (stock #003474).

Initial experiments were performed by injecting VSV-G/FIV-CMVntbeta-gal(7.5×10⁹ TU/ml) or LCMV/FIV-CMVntbeta-gal (2.9×10⁸ TU/ml) into thestriatum of adult C57BL6/J mice. Injections were done as described[Stein and Davidson, supra], using coordinates of 0.4 mm rostral and 2mm lateral to bregma, and 3.0 mm depth. Five microliters of vectorpreparation were injected at a speed of 500 mL per minute. For thisexperiment, 3 mice were injected with each vector, and were sacrificedat 3 weeks post-injection. In subsequent experiments, injectioncoordinates were adjusted to 1.0 mm rostral and 1.7 mm lateral to bregmaand 2.5 mm depth for adult mice, or 0.8 mm rostral and 1.3 mm lateral tobregma and 2.0 mm depth for 3-week old mice. For thenon-5-bromo-2′-deoxyuridine (BrdU) experiments, six 16-week old C57BL6/Jmice and four 3-week old C57BL6/J mice were injected withLCMV/FIV-CMVbeta-gal (6×10⁸ TU/ml; 9.1×10⁹ IntU/ml). Mice weresacrificed at either three weeks or 7.5 weeks post injection. For BrdUexperiments, three adult mice were injected with LCMV/FIV-CMVbeta-gal onday 0, and received intraperitoneal (i.p.) injections of BrdU (150mg/kg) daily for 18 days and were sacrificed 24 hours after the lastBrdU injection. The BrdU (Sigma-Aldrich Inc., St. Louis, Mo.) wasdissolved in 0.007 N NaOH/0.9% NaCl at 10 mg/ml. For experiments withROSA26 Cre reporter mice, three adult mice were injected withLCMV/FIV-GFAPcre (1.4×10⁹ IntU/ml), and sacrificed at three weekspost-injection.

At sacrifice, mice were anaesthetized and perfused with 2%paraformaldehyde in phosphate-buffered saline, pH 7.4 (PBS). Brains weredissected out and post-fixed overnight at 4° C., cryo-protected bysinking in 30% sucrose/PBS for 36 to 48 hours at 4° C. and embedded inOCT freezing compound (Sakura Finetek USA, Torrance, Calif.). Ten Tmcryosections were placed on glass-plus slides, and 40 Tm cryosections(floating) were placed in PBS.

X-gal and Immuno-fluorescent staining: For histological detection ofbeta-galactosidase enzymatic activity, 10 Tm cryosections on slides werestained with X-gal (Sigma-Aldrich) for 6 hours at 37° C. andcounterstained with neutral red.

For immuno-fluorescent staining, the antibodies used were as follows:rabbit anti-beta-galactosidase (BioDesign International, Saco, Me.)kit-conjugated to alexa 488 (Molecular Probes Inc., Eugene, Oreg.) andused at 5 Tg/ml; monoclonal mouse anti-NeuN (Chemicon InternationalInc., Temecula, Calif.) used at 1/200; Cy-3-conjugated monoclonal mouseanti-GFAP (Sigma) used at 1/5000; monoclonal mouse anti-class IIIbeta-tubulin (TuJ1 clone, R&D Systems, Minneapolis, Minn.) used at1/300; monoclonal rat anti-BrdU (Accurate Chemical & Scientific Corp.,Westbury, N.Y.) used at 1/500; and appropriate alexa 568-or alexa647-conjugated secondary antibodies from goat (Molecular Probes) used at1/500. Ten-micrometer sections on slides or 40-Tm floating sections werestained essentially as described [Stein and Davidson Meth. Enzymol.,346:433-454 (2002)]. Briefly, sections were blocked with 10% normal goatserum in PBS/0.2% triton-X-100 (PBS-T) for 1-2 hours at roomtemperature, stained with primary antibodies diluted in PBS-T at 4° C.overnight (10 Tm sections on slides) or for 48 hours (40-Tm floatingsections), washed with PBS and stained with secondary antibodies for 1hour (10 Tm sections) or 3 hours (40 Tm sections) at room temperature,washed and coverslipped with Vectashield (Vector Laboratories Inc.,Burlingame, Calif.). For BrdU staining, slides were blocked and stainedwith anti-beta-galalexa 488 overnight at 4° C., washed and acid-treatedwith 2N HCL in PBS-T for 30 minutes at 37° C., washed at roomtemperature with 0.1M sodium tetraborate (pH 8.5) followed by PBS, thenre-blocked for 1 hour and stained overnight with anti-BrdU and eitherTuJ1 antibody or anti-NeuN at 4° C., then washed and stained withsecondary antibodies as described. To estimate the number of BrdUpositive neurons in the OB, beta-gal positive cells were counted inmultiple OB fields in eight 10-Tm sections at 100 Tm intervals andscored as BrdU negative or positive.

Microscopic images were captured using a Spot RT camera and associatedsoftware (Diagnostic Instruments Inc., Sterling Heights, Mich.).Confocal images were captured on a Zeiss LSM 510 confocal microscopeusing LSM camera and software.

Pattern of Transgene Expression

A lentivirus vector pseudotyped with the LCMV WE-64 envelopeglycoprotein and carrying a nuclear-targeted bacterialbeta-galactosidase (ntbeta-gal) transgene driven by the cytomegalovirus(CMV) immediate early enhancer/promoter (LCMV/FIV-CMVntbeta-gal) wasinjected into the striatum of adult mice to assess the pattern of celltransduction in the brain. X-gal staining of para-sagittal sections atthree weeks postinjection indicated that significant numbers oftransgene-expressing cells were present not only in the striatum aroundthe injection site, but also in the OB. This was unlikeVSV-G-pseudotyped vector, which typically shows high level transductionin the striatum of predominantly neurons [Blomer et al., J. Virol.,71(9):6641-6649 (1997); Wong et al., Mol. Ther., 9:101-111 (2004); andBrooks et al. Proc. Natl. Acad. Sci. USA 99:6216-6221 (2002)], withrelatively few transgene-expressing cells found in the OB.

In a subsequent experiment, 3-and 16-week old mice were injected withLCMV/FIV-CMVbeta-gal. Here the beta-gal was cytoplasmically localized,rather than nuclear targeted, thus allowing discernment of cell types onthe basis of both morphology and confocal analysis afterimmuno-fluorescent staining for beta-gal and markers for astrocytes(GFAP), neurons (NeuN), or migratory neuroblasts (class IIIbeta-tubulin).

Using immuno-fluorescence beta-gal positive cells were detected in thestriatum, SVZ, RMS, and OB at 3 weeks post-injection. This pattern wasobserved for both age groups of mice, but the extent oftransgene-expressing cells was greater in the younger age group. Bymorphological criteria, most of transgene-expressing cells in theinjected area of the striatum appeared to be astrocytes, while those inthe OB appeared to be neurons.

Notably, many of the neurons extended a single unbranched apicaldendrite, characteristic of class 3 developing granule neurons describedby Petreanu and Alvarez-Buylla, J. Neurosci., 22:6106-6113 (2002).Confocal microscopy after dual immunofluorescent staining showed overlapof beta-gal and GFAP signals in the striatum, SVZ, and to a lesserextent in the proximal RMS, indicating that transgene-expressing cellsnear the injection site were predominantly astrocytes. In the horizontal(rostral) aspects of the RMS, overlap with GFAP was not apparent. Dualimmunofluorescent staining for beta-gal and class III betatubulin, amarker expressed early in neuronal commitment and prominent in migratoryneuroblasts, showed co-localization throughout the RMS, especially inthe rostral aspects. This was evident at both 3 weeks and 7.5 weeks postgene transfer. Moreover, the morphology of the beta-gal expressing cellsin the RMS was typical of migratory neuroblasts: elongated cells with aleading appendage and growth cone [Wichterle et al., Neuron 18:779-791(1997)]. In the OB, confocal analysis showed that the beta-galexpressing cells contained NeuN positive nuclei, confirming theiridentity as neurons. This pattern of transgene expression was highlysuggestive of transduction of SVZ neural progenitor cells that give riseto the neuronal precursors that migrate to and terminally differentiatein the OB.

BrdU Incorporation

To confirm that the OB neurons and their migratory precursors wereindeed the progeny of proliferating progenitor cells, the nucleotideanalog BrdU was injected into mice that had received unilateralLCMV/FIV-CMVbeta-gal injection in the brain striatum. BrdU was injectedi.p. daily from day 1 through day 18 relative to vector injection on day0, and mice were sacrificed 24 hours after the final BrdU dose. Confocalmicroscopy after triple staining for beta-gal, NeuN and BrdU, showedthat BrdU-labeled and non-labeled beta-gal-expressing neurons werepresent in the OB. Approximately 49% of the transgene-expressing neuronsin the OB were BrdU positive, while the rest were BrdU negative. SinceBrdU was injected only once per day, the BrdU negative cells may havearisen from mitotically active precursors that escaped BrdU labeling.Alternatively, the BrdU⁻/beta-gal⁺ neurons in the OB may have arisenfrom precursors that were transduced at post-mitotic state. With respectto the triple positive cells (beta-gal⁺/NeuN⁺/BrdU⁺), it is clear thatcell division occurred after LCMV-mediated vector uptake, and thus theseneurons in the OB were derived from a progenitor that underwent celldivision after transduction. Similarly, BrdU⁺/beta-gal⁺ migratoryneuroblasts were observed in the RMS, indicating the ongoing generationof neuroblasts from a mitotically active transduced stemcell/progenitor.

Type B Astrocytes are Transduced

The transduced cell type giving rise to the neuroblasts that traffic tothe OB could be either a slowly dividing neural stem cell and/or arapidly dividing progenitor cell. Doetsch and colleagues describe aGFAP-expressing type B astrocyte that has the properties of a neuralstem cell [Doetsch et al., Cell 97:703-716 (1999)]. To determine whetherthis cell type is a target of the LCMV-pseudotyped FIV, anLCMV-pseudotyped FIV vector was constructed carrying the cre recombinasetransgene under control of the GFAP promoter (LCMV/FIV-GFAPcre).LCMV/FIV-GFAPcre was injected unilaterally into the striatum of ROSA26Cre reporter mice, which harbor “loxP-stop-loxP-lacz” sequencesdownstream of an endogenous, ubiquitous promoter. If type B astrocytesin the SVZ were transduced, this should have resulted in expression ofcre recombinase and excision of the “stop” signals, leading toexpression of beta-gal in the transduced cells and their progeny. Thereporter mice were sacrificed 3 weeks after vector injection andpara-sagittal sections were analyzed by confocal microscopy afterimmuno-fluorescent staining. Beta-gal positive cells were detectedwithin the SVZ, RMS, and the OB of the LCMV/FIV-GFAPcre-injected mice.No beta-gal expressing cells were detected in the contralateral RMS orOB. Confocal analysis showed that beta-gal positive cells in the RMSwere also class III beta-tubulin positive, while those in the OBco-localized with NeuN. Since cre is driven off the GFAP promoter, thesecells must have descended from a transduced astrocyte. Together, theresults indicate that the LCMV-pseudotyped vector transduced type Bastrocytes, the leading candidate for neural stem cells in the SVZ.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method for transducing a neural progenitor cell with a transgene,comprising contacting the cell with a pseudotyped retrovirus virioncomprising a Lymphocytic Choriomeningitis Virus strain WE-54 envelopeglycoprotein and the transgene, wherein the cell is transduced with thetransgene.
 2. The method of claim 1, wherein the retrovirus virion is alentivirus virion.
 3. The method of claim 2, wherein the lentivirusvirion is a feline immunodeficiency virus virion.
 4. The method of claim1, wherein the transgene is a remedial gene.
 5. The method of claim 1,wherein the envelope glycoprotein comprises a phenylalanine at residue260, and wherein residue 260 is relative to the methionine at the firstposition of the sequence set forth in GenBank Accession No. AJ318512. 6.The method of claim 1, wherein the envelope glycoprotein comprises aphenylalanine at residue 153, and wherein residue 153 is relative to themethionine at the first position of the sequence set forth in GenBankAccession No. AJ318512.
 7. The method of claim 6, wherein the envelopeglycoprotein further comprises a phenylalanine at residue 260, andwherein residue 260 is relative to the methionine at the first positionof the sequence set forth in GenBank Accession No. AJ318512.